Screening and selection of most potent diazotrophic cyanobacterial isolate exhibiting natural tolerance to rice field herbicides for exploitation as biofertilizer

J. Basic Microbiol. 46 (2006) 3, 219–225 DOI: 10.1002/jobm.200510074

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0233-111X/06/0306-0219

(Algal Biotechnology Laboratory, Department of Biological Science, Rani Durgavati University, Jabalpur-482001, India)

Screening and selection of most potent diazotrophic cyanobacterial isolate exhibiting natural tolerance to rice field herbicides for exploitation as biofertilizer

SURENDRA SINGH and PALLAVI DATTA*

(Received 01 August 2005/Returned for modification 01 September 2005/Accepted 08 September 2005)

Periodic applications of heavy dosages of herbicides in modern rice-agriculture are a necessary evil for obtaining high crop productivity. Such herbicides are not only detrimental to weeds but biofertilizer strains of diazotrophic cyanobacteria also. It is therefore, essential to screen and select such biofertilizer strains of diazotrophic cyanobacteria exhibiting natural tolerance to common rice-field herbicides that can be further improved by mutational techniques to make biofertilizer technology a viable one. Therefore, efforts have been made to screen five dominant diazotrophic cyanobacterial forms e.g. filamentous heterocystous Nostoc punctiforme, Nostoc calcicola, Anabaena variabilis and unicellular Gloeocapsa sp. and Aphanocapsa sp. along with standard laboratory strain Nostoc muscorum ISU against increasing concentrations (0–100 mg l–1) of four commercial grade common rice-field herbicides i.e. Arozin, Butachlor, Alachlor and 2,4-D under diazotrophic growth conditions. The lethal and IGC50 concentrations for all four herbicides tested were found highest for A. variabilis as compared to other test cyanobacteria. The lowest reduction in chlorophyll a content, photosynthetic oxygen evolution, and N2-fixation was found in A. variabilis as compared to other rice field isolates and standard laboratory strain N. muscorum ISU. On the basis of prolong survival potential and lowest reductions in vital metabolic activities tested at IGC50 concentration of four herbicides, it is concluded that A. variabilis is the most potent and promising cyanobacterial isolate as compared with other forms. This could be further improved by mutational techniques for exploitation as most potential and viable biofertilizer strain.

Cyanobacteria (blue-green algae) are gram negative, oxygenic, photosynthetic prokaryotes. The dual capacity of fixing atmospheric nitrogen and carbon makes them attractive as a source of nitrogenous biofertilizer in rice agriculture (STEWART et al. 1987). In modern agricultural practices, uses of herbicides (pesticides) are an important factor for obtaining high crop productivity. Herbicides are reported to adversely affect the diversity and surviv-ability of cyanobacterial strains by inhibiting growth, macromolecular synthesis (VAISHAM-PAYAN et al. 1978, IRISARRI et al. 2001, KAUR et al. 2002), photosynthetic and nitrogenase activity (LEGANES and FERNANDEZ 1992, SINGH and TIWARI 1988), amino acid synthesis pathways (STEINRUCKEN and ARNHEIM 1980), mutagenic actions (SINGH et al. 1979, SINGH and VAISHAMPAYAN 1978) or even lead to complete elimination (KHAN and VAISHYA 1995). The extreme sensitivity of diazotrophic cyanobacteria to the toxicity of rice field herbicides is a major cause of concern for successful exploitation of cyanobacteria as biofer-tilizer. The present cyanobacterial biofertilizer technology is scientifically not very sound and reliable, as most of the desired features were not taken into consideration while prepar-ing inoculums. Thus it is essential to select and use such cyanobacterial biofertilizer strains capable of tolerating or resisting toxic actions of herbicides under field conditions. Agro-climatic conditions of any region play an important role in the establishment and productive

* Corresponding author: Dr. P. DATTA; e-mail: [email protected]

220 S. SINGH and P. DATTA

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

performance of microbial inoculants (GOYAL 1997). Therefore, screening and selection of naturally occurring dominant forms of N2-fixing cyanobacteria seemingly better adapted for a particular agro-ecosystem are bound to play a very significant role in improving soil fertil-ity and crop productivity upon algalization. Efforts have been made to screen the biological responses of five dominant diazotrophic cyanobacteria out of several isolates from the rice-field and one standard laboratory strain Nostoc muscorum ISU against four common rice field herbicides, Arozin, Alachlor, Butachlor and 2,4-D. The isolates exhibited differential tolerance towards these herbicides. Among them, the heterocystous diazotrophic cyanobac-terium Anabaena variabilis was ecophysiologically found most tolerant towards all four herbicides tested. Hence this potential and better adapted strain can be further analyzed and improved for biotechnological exploitation as beneficial biofertilizer strain in improving soil health and agricultural output.

Materials and methods

Source of organisms and growth conditions: The filamentous N2-fixing cyanobacteria Nostoc punctiforme, Nostoc calcicola, Anabaena variabilis and the unicellular N2-fixing Gloeocapsa sp. and Aphanocapsa sp. used in the present investigation were isolated from rice fields (SINGH et al. 2000), whereas standard laboratory strain Nostoc muscorum ISU (ATCC 27893) was obtained from Prof. A. K. KASHYAP, Department of Botany, Banaras Hindu University, Varanasi, India. Cultures were axenically grown in BG11 medium (RIPPKA et al. 1979) devoid of any combined nitrogen source (hereafter called as N2-medium). Cultures were incubated in an air-conditioned culture room main-tained at 25 ± 1 °C and illuminated with cool day fluorescent lights (Photon flux density 45 µEm–2 s–1) under 18 h light and 6 h dark cycle.

Herbicides: All the herbicides used were of commercial grade. Arozin (30 EC) was obtained from Agr. Evo. Ltd. (Ankleshwar, India), Alachlor (45.1 EC) and Butachlor (93.34 EC) from Evid and Co. Pesticides Pvt. Ltd. (Ankleshwar, India) and 2,4-D Ethyl ester (38 EC) from Monsanto Chemicals of India Ltd. (Mumbai, India). Different concentrations of the respective herbicides were prepared by appropriate dilution (according to EC) in pre-cooled sterilized double distilled water and were filter sterilized through millipore membrane filter.

Determination of growth, photosynthesis and nitrogenase activity of cyanobacterial isolates: Impact of increasing concentrations (0–100 mg l–1) of herbicides Arozin, Alachlor, Butachlor and 2,4-D on the growth and survival of diazotrophic cyanobacterial isolates were determined in N2-medium by monitoring variations in the concentrations of chlorophyll a pigments (MACKINNEY 1941) at regular intervals of 24 h as a parameter of growth. Growth was also measured in terms of whole cell protein content following the method of LOWRY et al. (1951). For all the experiments, exponentially growing cells (6 d old) were harvested by centrifugation (3000 × g, 5 min), washed thrice with sterilized double distilled water and dispensed equally in assay flasks. Cultures were incubated under photo-autotrophic growth conditions as described above. The cultures without addition of herbicides were taken as control. The 50% inhibitory growth concentrations for the isolates were determined by monitoring survivability of cells on plates containing graded concentrations of herbicides. The percentage survival of the isolates were calculated by the following method:

×

Number of colonies on the herbicide treated plates100

Number of colonies on the untreated control plates.

The concentration of the herbicide at which 50% of colonies survived as compared to untreated control culture was termed as IGC50 and the concentration at which no colonies survived (complete lysis) were considered the lethal concentration. The experiment was repeated thrice and every time five sets of plates were taken for each herbicide concentration. The data were analyzed by using a statistical computer program by determining means and standard errors.

Diazotrophic cyanobacteria and herbicides 221


Photosynthetic O2 evolution was measured using CLARK type Oxygen Electrode (Hansatech Instrument Ltd, England) as described by RAO et al. (1984). N2-fixing abilities of test cyanobacterial strains under herbicides stress were determined Gas Chromatographically (CIC, India) by acetylene reduction assay (ARA) following the method of STEWART et al. (1967). All the experiments were repeated thrice before arriving to conclusion. The results were expressed as mean values of experiments ± standard errors. All the chemicals used were of Analytical grade.

Results

Diazotrophic cyanobacterial isolates showed gradual inhibition in the growth with increas-ing dosage of herbicides. Complete lysis of the cultures occurred within 8–12 days of incu-bation. The lethal and IGC50 concentrations recorded for the cyanobacterial isolates are given in Tables 1 and 2. Growth measured in terms of chlorophyll a indicated maximum inhibition (53%–65% of control) in N. muscorum followed by Aphanocapsa sp. and Gloeocapsa sp. i.e. nearly dou-ble as compared to A. variabilis at IGC50 by the end of 8th d of incubation (Fig. 1). The ini-tial chlorophyll a (µg ml–1) 0.24 in N. muscorum was increased to 1.61 in untreated control culture, whereas 0.56, 0.75, 0.75, 0.69 chlorophyll a in cells treated with IGC50 of Arozin, Alachlor, Butachlor and 2,4-D respectively was recorded on 8th d of growth. Simultaneously growth was also determined by estimating protein content (data not shown). Substantial inhibition in photosynthetic activity of cyanobacterial isolates was recorded by the end of 6th d of growth. Highest inhibition (26%–70% of control) was observed in N. muscorum and minimum (7%–36% of control) in A. variabilis at IGC50. Gloeocapsa sp. and Aphanocapsa sp. followed almost similar pattern of inhibition as in N. muscorum (Fig. 2). The initial O2-evolution 19.0 µ mol O2 mg–1 chl a min–1 in N. muscorum was

Table 1 IGC50 values (mg l–1) of herbicides for cyanobacterial isolates

Cyanobacterial Isolates Arozin Alachlor Butachlor 2,4-D

1. Nostocmuscorum ISU 4.9 ± 0.15 15.0 ± 0.07 10.4 ± 0.39 10.5 ± 0.69 2. Nostoc punctiforme 4.7 ± 0.31 15.0 ± 0.08 14.8 ± 0.27 9.9 ± 0.09 3. Nostoc calcicola 5.0 ± 0.06 14.8 ± 0.49 9.7 ± 0.45 9.7 ± 0.48 4. Anabaena variabilis 9.9 ± 0.24 15.4 ± 0.36 15.0 ± 0.17 14.8 ± 0.48 5. Gloeocapsa sp. 5.0 ± 0.17 15.2 ± 0.07 10.0 ± 0.08 5.0 ± 0.09 6. Aphanocapsa sp. 5.1 ± 0.10 14.8 ± 0.35 10.3 ± 0.32 5.0 ± 0.18

The data is an average (±SEM) of three independent experiments

Table 2 Lethal concentrations (mg l–1) of herbicides for cyanobacterial isolates

Cyanobacterial Isolates Arozin Alachlor Butachlor 2,4-D

1. Nostocmuscorum ISU 15.0 ± 0.21 20.0 ± 0.42 15.0 ± 0.45 15.0 ± 0.35 2. Nostoc punctiforme 20.0 ± 0.32 25.0 ± 0.25 25.0 ± 0.38 25.0 ± 0.32 3. Nostoc calcicola 20.0 ± 0.17 25.0 ± 0.15 20.0 ± 0.26 25.0 ± 0.17 4. Anabaena variabilis 25.0 ± 0.31 25.0 ± 0.21 25.0 ± 0.16 25.0 ± 0.39 5. Gloeocapsa sp. 15.0 ± 0.17 20.0 ± 0.17 25.0 ± 0.15 15.0 ± 0.48 6. Aphanocapsa sp. 10.0 ± 0.18 20.0 ± 0.18 20.0 ± 0.22 10.0 ± 0.45

The data is an average (±SEM) of three independent experiments



0

0.5

1

1.5

2

2.5

Control Arozin Alachlor Butachlor 2.4-D

Chl

orop

hyll

a (

µg m

l-1)

Nostoc muscorum Anabaena variabilis Nostoc puntiforme

Nostoc calcicola Gloeocapsa sp. Aphanocapsa sp. Fig. 1 Effect of IGC50 concentration of herbicides on chlorophyll a content of cyanobacterial isolates. Error bars represent standard errors

0

5

10

15

20

25

30

35


Pho

tosy

nthe

sis

(µm

ol.O

2m

g-1ch

la.m

in-1

)

Nostoc muscorum Anabaena variabilis Nostoc punctiforme

Nostoc calcicola Gloeocapsa sp. Aphanocapsa sp. Fig. 2 Effect of IGC50 concentration of herbicides on photosynthesis in cyanobacterial isolates. Error bars represent standard errors significantly reduced to 8.0, 15.0, 19.0, 15.0 where as 18.0, 22.0, 24.0, 26.0 was recorded for A. variabilis from initial rate of 22.0 in 2,4-D, Arozin, Butachlor and Alachlor treated cultures respectively at IGC50 on 6th d of growth. Significant inhibition in nitrogenase activity was observed in herbicide treated cultures after 48 h of incubation. Among the isolates A. variabilis exhibited 22%–33% inhibition of control whereas nearly 2 fold increase in reduction was observed in Aphanocapsa sp. Gloeocapsa sp. and N. muscorum at IGC50 (Fig. 3). The initial nitrogenase activity of 0.44 n mol C2H4 mg–1 chl a h–1 was reduced to 0.18, 0.20, 0.22, 0.24 in N. muscorum whereas 0.33, 0.35, 0.37, 0.38 was recorded in A. variabilis treated with 2,4-D, Arozin, Butachlor and Alachlor treated cultures respectively at IGC50 after 48 h of incubation.



0

0.1

0.2

0.3

0.4

0.5

0.6


N2

fixa

tion

(nm

ol.C

2H4.

µg-1

chla

h-1)

Nostoc muscorum Anabaena variabilis Nostoc punctiforme

Nostoc calcicola Gloeocapsa sp. Aphanocapsa sp. Fig. 3 Effect of IGC50 concentration of herbicides on N2 fixation in cyanobacterial isolates. Error bars represent standard errors

Discussion

The results indicate variation in the relative tolerance level of the diazotrophic cyanobacte-rial strains to the growth toxic effects of the four herbicides tested. Variations in the relative tolerance potentiality of the test cyanobacterial isolates to the growth toxic concentration of the herbicides, seems to result from interactions between mode of herbicidal actions with morphological, physiological, biochemical and genetic properties of cyanobacteria. Herbi-cides have been shown to have differential effects on various vital metabolic activities and the tolerance level of the each strain differs with each other depending upon the species, kind of herbicides and chemical formulations (POWELL et al. 1991, ANAND and SUBRAMA-NIAN 1997, FAIRCHILD et al. 1998). In the present study, chlorophyll a synthesis was found to be severely affected in Arozin treated cultures of N. muscorum, Gloeocapsa sp. and Aphanocapsa sp. as compared to A. variabilis, N. punctiforme and N. calcicola (Fig. 1) and complete lysis of the cultures occurred between 10–25 mg l–1 in different isolates. On contrary Arozin up to 10 mg l–1 was reported to enhance chlorophyll a synthesis and nitrogenase activity in A. variabilis ARM 310 by GOYAL et al. (1991). Both photosynthetic and nitrogenase activity exhibited maximum inhibition at IGC50 concentration of 2,4-D treated culture of N. muscorum ISU followed by Gloeocapsa sp. and Aphanocapsa sp. (Figs. 2 and 3). However, such reduction in photosynthetic and nitro-genase activities in another Gloeocapsa sp. and Anabaena UAM 202 was found at much higher concentration i.e. 175 mg l–1 (TOZUM and SIVACI 1993) and 10 mM of 2,4-D (LEGA-NES and FERNANDEZ 1992) respectively. N. muscorum ISU, Gloeocapsa sp. and Aphanocapsa sp. also exhibited substantial inhibi-tion in growth, photosynthetic and nitrogenase activities as compared to A. variabilis, N. punctiforme and N. calcicola at IGC50 concentration (9.7 mg l–1–15.4 mg l–1) of Buta-chlor and Alachlor (Figs. 1, 2 and 3). However, on contrary only partial inhibition in growth and photosynthesis at 100 mg l–1 of Butachlor in another isolate of Gloeocapsa sp. was observed by SINGH et al. (1986). Similarly LIKHITKAR and TARAR (1996) reported partial inhibition in growth and nitrogenase activity at 200 mg l–1 of Butachlor in N. commune and N. muscorum.



Complete inhibition in growth and nitrogenase activity was reported at 80 mg l–1 of Alachlor in A. doliolum, N. muscorum and Aphanothece stagnina (SINGH et al. 1978), which is comparatively high as compared to 20–25 mg l–1, recorded in our test cyanobacterial isolates. Ongoing discussion thus suggests that diazotrophic cyanobacterial strains isolated from diverse agro-climatic zones possesses different degrees of inherent natural tolerance towards various class of herbicides. The present finding shows maximum reduction in growth, photosynthesis and nitrogenase activity in N. muscorum ISU a laboratory strain not exposed to rice-field conditions and the lowest in A. variabilis a field isolate at IGC50 concentrations of all four herbicides tested. This indicates that A. variabilis is the most tolerant to all four herbicides and could serve as an ideal cyanobacterial biofertilizer inoculant if exploited further to improve soil fertility and crop productivity. The results further suggest the presence of different degree of inher-ent natural tolerance among the strains isolated from same agro-ecosystem. Thus to develop a viable cyanobacterial biofertilizer technology, region specific potential diazotrophic cyanobacterial strains should be isolated, screened and improved for utilization as potential biofertilizer strain to improve soil fertility, soil health and crop productivity.

Acknowledgements

We are thankful to the Head, Department of Biological Science, R. D. University, Jabalpur (M.P.), India for facilities and U.G.C., New Delhi for financial assistance.

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Mailing address: Dr. PALLAVI DATTA, Algal Biotechnology Laboratory, Department of Biological Science, Rani Durgavati University, Jabalpur-482001, India E-mail: [email protected]

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Screening and selection of most potent diazotrophic cyanobacterial isolate exhibiting natural tolerance to rice field herbicides for exploitation as biofertilizer