Isolation and Characterization of a Multiple Herbicide Resistant

Indian Journal of Biotechnology Vol 11, January 2012, pp 77-85

Isolation and characterization of a multiple herbicide resistant strain [Av(MHR)Ar,Al,B,D] of diazotrophic cyanobacterium Anabaena variabilis Surendra Singh, Pallavi Datta* and Archna Tirkey Algal Biotechnology Laboratory, Department of Biological Sciences, Rani Durgavati University, Jabalpur 482 001, India Received 1 August 2010; revised 18 December 2010; accepted 20 February 2011 The present study was aimed to isolate a multiple herbicide resistant strain [(MHR)Ar,Al,B,D] of Anabaena variabilis exhibiting resistance against four common rice-field herbicides, viz., Arozin, Alachlor, Butachlor and 2,4-D. The multiple herbicide resistant (MHR) strain was isolated by spontaneous mutational techniques. Of the four spontaneously occurring mutants, Arozin resistant mutant (Ar-R) exhibited 92-98% survival in lethal concentrations of Arozin, Alachlor, Butachlor and 2,4-D, and was designated as MHR strain, Av(MHR)Ar,Al,B,D. The MHR strain exhibited faster growth rate, substantial increase in macromolecular contents, photosynthesis, nitrogenase and glutamine synthetase (GS) activity not only in graded concentration of herbicides but below (0-25 mg L-1) and above the lethal dosages (25-80 mg L-1), and even in the presence of combined lethal dosages of all four herbicides as compared to wild type as well as Alachlor, Butachlor and 2,4-D resistant strains. The cross resistance relationship in Arozin resistant (Ar-R) mutant strain of A. variabilis with lethal dosages of Alachlor, Butachlor and 2,4-D did indicate that it was a single mutational event and resistance to Arozin was associated with the acquisition of resistance to Alachlor, Butachlor and 2,4-D. Evidently such improved Av(MHR)Ar,Al,B,D strain of diazotrophic cyanobacterium A. variabilis under herbicide(s) stressed agro-ecosystem, particularly rice-fields, would likely to serve as efficient biofertilizer. Keywords: Biofertilizer, cyanobacteria, herbicides, mutant

Introduction Some reports are available on genetic improvement Diazotrophic cyanobacteria (blue green algae) have in certain cyanobacteria through mutagenesis been implicated in sustaining the cultivation of conferring herbicide (Norflurazon, Fluridone, wetland rice for centuries because of their inherent Dinoseb, Amitrole) resistance to Synechococcus sp. ability to add fixed nitrogen to such habitats under PCC 7942 and Synechocystis sp. PCC 68039-11. aerobic photosynthetic conditions1-3. This has led to Gloeocapsa sp., a natural isolate, showing resistance the development of biofertilizer technology involving against two rice field herbicides, Machete (Butachlor) inoculation of the paddy fields with suitable and Basalin12 and Nostoc muscorum exhibiting diazotrophic cyanobacterial strains4-7. In recent years resistance against Amitrole and Carbendazine13 have the practice of using diazotrophic cyanobacteria as an been isolated. Evidently most of the work has been efficient source of nitrogenous biofertilizer for rice concentrated on isolation of cyanobacterial mutants crops has been advocated and adapted in many through mutagenesis exhibiting resistance either developing countries8. However, the productivity of against one or two herbicides. Hence, there is a need the wetland rice agriculture is heavily dependent on to develop more and more herbicide resistant the periodic and extensive use of highly toxic diazotrophic cyanobacterial strains native to rice field herbicides for selective elimination of weeds of rice ecosystem by mutational techniques conferring crops. The extreme sensitivity of cyanobacteria to resistance to a number of rice-field herbicides. The rice-field herbicides is limiting the wide utilization of concept of developing multiple herbicide resistant cyanobacterial biofertilizer technology. A successful strains of diazotrophic cyanobacteria is important biotechnology involving the use of N2-fixing since combination of herbicides are applied in modern cyanobacteria as biofertilizer in modern rice rice agriculture and the residual toxic effects of these agriculture would require them to have the additive agrochemicals persists in the environment14,15. property of resistance to various rice field herbicides. Therefore, efforts have been made to develop a ______multiple herbicide resistant strain (MHR) of a natural *Author for correspondence: Tel: +91-9300126788; Fax: +91-761-603752. diazotrophic cyanobacterial isolate Anabaena E-mail: [email protected] variabilis by isolating and examining cross resistant 78 INDIAN J BIOTECHNOL, JANUARY 2012

relationship among the spontaneous mutant clones conditions as mentioned earlier. After 3-4 wk, exhibiting resistance against four common rice-field cross-resistant colonies appearing on the surface of herbicides, viz., Arozin, Alachlor, Butachlor and agar plates were picked up and transferred to sterile 2,4-D. Arozin resistant mutant of A. variabilis exhibited N2-medium for the growth. Among the four herbicide cross resistance against lethal dosages of Alachlor, resistant mutants tested, Arozin resistant mutant Butachlor and 2,4-D. This multiple herbicide resistant (Ar-R) clones survived in the lethal concentrations of mutant exhibited enhanced growth, survivability, Alachlor, Butachlor and 2,4-D. This mutant was N2-fixation, photosynthesis and other vital metabolic considered to be cross resistant and was designated as activities as compared to wild type strain. MHR strain [Av(MHR)Ar,Al,B,D]. Butachlor (B-R), Alachlor (Al-R) and 2,4-D resistant (D-R) strains Materials and Methods could not survive in lethal dosages of other Organism and Growhth Conditions herbicides. For spontaneous reversion studies, The axenic clonal culture of N2-fixing exponentially growing culture of MHR strain was cyanobacterium A. variabilis, a rice field isolate16, harvested by repeated centrifugation (5000× g; 17 was routinely grown in BG11 medium devoid of any 5 min) and washed with sterile double distilled water combined nitrogen source (N2-medium). Cultures and resuspended in fresh N2-medium. Inoculum 5 were incubated for the growth in an air-conditioned (3 × 10 CFU) was plated on solid N2-medium with or culture room maintained at 25±1°C fitted with cool without herbicide treatment and incubated for 15 d day fluorescent light. Photon flux density of light on under photo autotrophic growth conditions for scoring the surface of the vessel was 45 µE m-2 s-1 for 18 h d-1. revertants. The same experiment was repeated thrice. No change in the colonial growth in both the Mutant Isolation treatment was recorded. A. variabilis did not survive beyond a concentration -1 Characterization of MHR Strain in Graded Concentrations of of 25 mg L of Arozin, Alachlor, Butachlor and Herbicides 2,4-D. Accordingly, its diazotrophically grown The MHR strain was characterized in terms of 7 cultures (5.0 × 10 CFU) were seeded per diazotrophic following parameters: nutrient plate containing 50 mg L-1 Arozin, 75 mg L-1 -1 -1 Determination of Growth Kinetics Butachlor, 75 mg L 2,4-D and 100 mg L Alachlor Growth kinetics of MHR strain along with wild to select out spontaneously occurring herbicide type A. variabilis was determined in diazotrophic resistant mutant clones. Colonies of the mutant growth conditions by estimating changes in appearing on the respective nutrient plates containing chlorophyll a and total protein content at regular herbicide were tested 3-4 times for their stability by interval of 24 h19,20 up to 12 d of growth. Generation streaking them on fresh nutrient plates containing time and specific growth rate was calculated using the same concentration of herbicides. Stable Arozin following formula: (Ar-R), Butachlor (B-R), 2,4-D (D-R) and Alachlor resistant (Al-R) mutant clones thus obtained were Kt = log10 (Nt/N0) grown and maintained in N2-medium along with their Where, K = Growth rate constant, t = Growth period, wild type strains under photoautotrophic growth Nt = Absorbance at time t, No = Absorbance at time 0. conditions as described above. 24 Generation time = Examination of Cross Resistant Relationship among Various Specific growth rate cons tan t Herbicide Resistant Mutant Strains of A. variabilis and Isolation of Multiple Herbicide Resistant (MHR) Mutant Determination of Macromolecular Contents Strain [Av (MHR)Ar,Al,B,D] Macromolecular contents, i.e., chlorophyll a, To establish cross resistant relationship between protein, carotenoids, phycocyanin, phycoerythrin, Arozin, Alachlor, Butachlor and 2,4-D resistant carbohydrate, DNA and RNA were determined at strains of A. variabilis, exponentially grown (6-d-old) regular intervals of 48 h of diazotrophic growth19-24. cultures of each resistant strain was plated on solid agar (2.0% w/v) based N2-medium containing lethal Determination of Heterocyst Frequency concentrations18 of all four herbicides individually. Heterocyst frequency of exponentially grown The plates were incubated in photoautotrophic growth diazotrophic culture of both MHR and wild type SINGH et al: ISOLATION AND CHARACTERIZATION OF A MULTIPLE HERBICIDE RESISTANT STRAIN 79

strains was determined microscopically and expressed Results in percentage as total number of heterocysts occurring The N2-fixing culture of wild type A. variabilis per 100 vegetative cells. exhibited a decrease in survival with increase in the concentrations of herbicides and reached almost a -1 Determination of Photosynthetic O2 Evolution and Dark O2 zero percent survival in 25 mg L of Arozin, Uptake Butachlor, Alachlor and 2-4,D. Spontaneous mutants The photosynthetic O2 evolution and respiratory O2 resistant to growth inhibitory action of Arozin, uptake of both parent and mutant strain was measured 25 Alachlor, Butachlor and 2,4-D were separately with Clark type oxygen electrode (Hansatech, U.K.) . isolated and the frequency with which each one of them arose was ~3 × 10-7. It was observed that among Measurement of Nitrogenase Activity the herbicide resistant mutant strains, Arozin resistant In vivo nitrogenase activity was measured by Gas mutant strain of A. variabilis (Ar-R) exhibited 99, 95, Chromatography (CIC, India) by acetylene reduction 26 94 and 92% survivability in the lethal dosages of assay technique . Arozin, Alachlor, Butachlor and 2,4-D, respectively;

whereas 100% sensitivity was exhibited by Alachlor, Determination of Glutamine Synthetase (GS) Activity In vivo Mn2+ dependent γ-glutamyl transferase Butachlor and 2,4-D resistant mutants when tested for activity of GS enzyme was measured27. cross resistance relationship. Thus, Arozin resistant mutant strain of A. variabilis showed cross resistance Performance of MHR strain Exposed to Lethal to Alachlor, Butachlor and 2,4-D and was designated Concentrations of Herbicides as multiple herbicide resistant mutant (MHR) isolate, Ar,Al,B,D Growth, macromolecular contents, heterocyst Av(MHR) . The reversion of MHR mutant was frequency, photosynthetic O2 evolution, respiratory tested by repeatedly growing the cells first without O2 uptake, nitrogenase and GS activity of both MHR herbicides, followed by with lethal concentrations of and wild type strain was compared under diazotrophic herbicides for several generations and was found to be growth conditions by exposing them to the lethal the stable mutant. The resistance level of MHR concentration of all the four herbicides applied mutant isolate was several times higher than the -1 together following the analytical methods as recommended field dosages (Arozin-0.098-0.15 mg L ; -1 described above. Alachlor-0.55-1.47 mg L ; Butachlor-0.368-1.47 mg -1 -1 L and 2,4-D-0.092-2.72 mg L ) of these herbicides. Herbicides This clearly demonstrates the biofertilizer potentials All the herbicides, viz., Arozin (30 EC) {S [N of such mutationally improved strain which will (4-chloro-phenyl-)-N-isopropyl-carbamoylmethyl-]- continue to survive and fix atmospheric nitrogen in O,O,dimethyl-dithiophosphate} (Agrevo Ltd., herbicide-treated agro ecosystem. Ankleshwar, India), Alachlor (45.1 EC) {2-chloro- The growth kinetics of wild type, herbicide 2’,6’-diethyl-N-(Methoxy methyl)acetanilide}, resistant strains and multiple herbicide mutant of Butachlor (93.34 EC) {N-(butoxy methyl)-2-chloro- A. varaibilis was compared under diazotrophic growth 2’,6’-diethyl acetanilide} (Evid & Co. Pesticides Pvt. conditions (Fig. 1). The specific growth rate constant Ltd., Ankleshwar, India) and 2,4-D ethyl ester calculated for wild type, Av(M)Al, Av(M)B, Av(M)D (38 EC) {4-(2,4-dichlorophenoxy)ethyl ester} and Av-MHR was 1.07, 1.14, 0.72, 1.13 and 1.23, and (Monsanto Chemicals of India Ltd., Mumbai, India), doubling time (G) was 22.4, 21, 33, 21.2 and 19.5 h, used were of commercial grade. Different respectively. Evidently, MHR was growing faster concentrations of the respective herbicides were than wild type as well as Alachlor resistant, Butachlor prepared by appropriate dilution (according to EC) in resistant and 2,4-D resistant strains of A. variabilis. pre-cooled sterilized double distilled water and were Under unstressed (control) condition the filter sterilized through Millipore membrane filter diazotrophic grown culture of Av(M)Al , Av(M)D and (0.22 µm). Av-MHR exhibited, respectively, 45, 48 and 59% increase in chlorophyll a (Table 1); 22, 22 and 23% in Statistical Analysis phycocyanin; 19, 23 and 25% in phycoerythrin; 17, The data were the mean of three independent 21 and 31% in protein; 16, 20 and 28% in experiments and expressed as ±SE (Standard Error). carbohydrate; 30, 36 and 42% in DNA; 15, 21 and 80 INDIAN J BIOTECHNOL, JANUARY 2012

32% in RNA (data not shown); 38, 38 and 48% in wild type strain exhibited 17% increase in γ-glutamyl photosynthetic O2 evolution (Table 2); 33, 36 and transferase activity of glutamine synthetase (GS) Al D 38% in dark O2 uptake (data not shown); 75, 75 and enzyme compared to that of Av(M) , Av(M) and Av- 88% in nitrogenase activity (Table 3) as compared to MHR strain (Table 4). Unlike others, the Butachlor wild type strain by the end of 6th d of diazotrophic resistant mutant [Av(M)B] showed reduction in growth. The heterocyst frequency was found pigment and other macromolecular contents than wild equivalent (7%) in the mutants and the wild type. The type strain. Chlorophyll a was reduced to 50% (Table 1); phycocyanin and phycoerythrin to 21 and 24%, respectively; protein to 58%; carbohydrate to 42%; DNA and RNA was reduced to 23 and 24%, respectively (data not shown), whereas photosynthetic O2 evolution and nitrogenase activity was found almost equivalent to wild type strain by the end of 6th d of diazotrophic growth (Tables 2 & 3). The heterocyst could not be detected in this mutant because of the drastic morphological changes. This could be due to the mutagenic property of Butachlor as reported earlier32. When the wild type and the mutants were exposed -1 Fig. 1—Diazotrophic growth of wild type and herbicide resistant to the graded concentration (0-100 mg L ) of mutants of A. variabilis herbicides, the wild type strain (at 15 mg L-1)

Table 1—Impact of graded concentration of herbicides on chlorophyll a content (µg mL-1) of wild type and herbicide resistant mutant strains of A. Variabilis

Cyanobacterial Herbicide strains (mg L-1) 0.0 10.0 15.0 25.0 50.0 85.0 100.0

Arozin Av (P) 1.2±0.1 0.6±0.1 0.5±0.1 0.1±0.03 ND ND ND Av (M)Ar-MHR 2.9±0.5 2.8±0.5 2.7±0.2 2.5±0.10 2.4±0.2 1.0±0.2 0.8±0.3 Av (M)Al 2.2±0.6 1.3±0.2 1.1±0.4 0.6±0.10 ND ND ND Av (M)B 0.6±0.5 0.5±0.2 0.3±0.5 0.1±0.04 ND ND ND Av (M)D 2.3±0.5 1.5±0.5 1.0±0.1 0.2±0.10 ND ND ND

Alachlor Av (P) 1.2±0.1 1.1±0.4 0.6±0.3 0.1±0.05 ND ND ND Av (M)Ar-MHR 2.9±0.5 2.9±0.3 2.9±0.2 2.8±0.60 2.6±0.6 2.0±0.5 1.0±0.1 Av (M)Al 2.2±0.6 2.2±0.2 2.1±0.5 2.1±0.60 1.0±0.1 0.8±0.2 0.7±0.2 Av (M)B 0.6±0.5 0.5±0.1 0.3±0.3 0.1±0.06 ND ND ND Av (M)D 2.3±0.5 1.3±0.2 1.2±0.3 1.0±0.10 ND ND ND

Butachlor Av (P) 1.2±0.1 0.9±0.1 0.5±0.1 0.2±0.1 ND ND ND Av (M)Ar-MHR 2.9±0.5 2.8±0.2 2.8±0.7 2.7±0.8 2.5±0.7 1.5±0.4 0.9±0.2 Av (M)Al 2.2±0.6 1.3±0.2 1.1±0.2 0.6±0.2 ND ND ND Av (M)B 0.6±0.5 0.6±0.1 0.6±0.1 0.6±0.2 0.3±0.1 0.2±0.1 0.1±0.04 Av (M)D 2.3±0.5 1.2±0.4 1.1±0.1 0.8±0.3 ND ND ND

2,4-D Av (P) 1.2±0.1 0.8±0.6 0.7±0.40 0.1±0.03 ND ND ND Av (M)Ar-MHR 2.9±0.5 2.8±0.9 2.7±0.60 2.6±0.80 2.5±0.7 1.1±0.5 0.8±0.4 Av (M)Al 2.2±0.6 1.5±0.5 1.4±0.04 0.5±0.10 ND ND ND Av (M)B 0.6±0.5 0.5±0.1 0.3±0.01 0.1±0.02 ND ND ND Av (M)D 2.3±0.5 2.3±0.9 2.3±0.80 2.2±0.70 1.0±0.5 0.7±0.2 0.4±0.1

The data are expressed as mean±SE ND: Not detectable SINGH et al: ISOLATION AND CHARACTERIZATION OF A MULTIPLE HERBICIDE RESISTANT STRAIN 81

-1 -1 Table 2—Impact of graded concentration of herbicides on photosynthetic O2 evolution (µmol O2 mg chl a min ) of wild type and herbicide resistant mutant strains of A. variabilis

Cyanobacterial Herbicide strains (mg L-1) 0.0 10.0 15.0 25.0 50.0 85.0 100.0

Arozin Av (P) 26.0±2.4 18.0±3.0 12.0±2.1 2.8±1.0 ND ND ND Av (M)Ar-MHR 50.0±3.5 49.8±2.5 49.7±3.9 49.6±4.0 49.2±2.5 31.5±2.2 25.0±3.1 Av (M)Al 42.0±3.2 24.3±2.4 18.2±2.0 5.0±1.2 ND ND ND Av (M)B 25.0±1.8 18.0±1.6 14.4±1.8 3.0±1.8 ND ND ND Av (M)D 42.0±2.0 25.8±4.0 20.3±3.2 5.2±4.1 ND ND ND

Alachlor Av (P) 26.0±2.4 21.0±1.8 12.0±1.5 3.2±1.0 ND ND ND Av (M)Ar-MHR 50.0±3.5 50.0±4.5 50.0±4.2 49.8±5.0 49.3±2.8 35.0±3.6 24.5±3.8 Av (M)Al 42.0±3.2 41.4±5.0 41.2±5.6 40.0±3.9 18.5±3.7 16.2±2.1 10.0±1.8 Av (M)B 25.0±1.8 19.5±2.9 16.2±3.0 5.4±2.5 ND ND ND Av (M)D 42.0±2.0 26.2±3.9 22.3±4.0 6.3±2.5 ND ND ND

Butachlor Av (P) 26.0±2.4 19.6±3.6 15.4±3.8 2.7±1.9 ND ND ND Av (M)Ar-MHR 50.0±3.5 49.9±2.4 49.8±4.0 49.7±5.0 49.2±3.5 33.5±2.4 24.5±2.3 Av (M)Al 42.0±3.2 27.0±1.2 26.0±2.1 6.0±1.6 ND ND ND Av (M)B 25.0±1.8 25.0±3.2 24.6±3.8 24.0±4.3 17.0±2.1 12.5±2.0 9.4±1.5 Av (M)D 42.0±2.0 25.0±2.6 25.5±2.7 5.4±2.6 ND ND ND

2,4-D Av (P) 26.0±2.4 17.0±1.6 10.0±2.3 2.0±1.0 ND ND ND Av (M)Ar-MHR 50.0±3.5 49.8±4.3 49.6±4.7 49.5±4.8 49.2±5.0 32.0±3.6 22.5±3.2 Av (M)Al 42.0±3.2 23.2±2.6 18.0±2.0 4.9±1.2 ND ND ND Av (M)B 25.0±1.8 17.5±3.4 14.0±3.0 2.7±1.6 ND ND ND Av (M)D 42.0±2.0 42.0±4.3 41.4±4.0 41.0±3.5 17.2±2.3 14.3±2.6 8.6±1.5

The data are expressed as mean±SE ND: Not detectable

exhibited 42-58% inhibition in chlorophyll a, 41-61% Alachlor and Butachlor; whereas no such reduction in photosynthesis, 75-95% in nitrogenase, 21-34% in was observed in case of Av-MHR (Tables 1-4). GS (transferase) activity and 70-80% in heterocyst Wild type strain lysed at 25 mg L-1, whereas frequency as compared to untreated control culture in Alachlor, Butachlor and 2,4-D resistant mutants response to all the four herbicides tested. The Av(M)Al exhibited complete growth inhibition at 50 mg L-1 of mutant showed 36-50% inhibition in chlorophyll a, herbicides of all four herbicides tested on 8 d of 38-57% in photosynthesis, 62-69% in nitrogenase, growth (Tables 1-4). Strikingly, no such inhibition 16-20% in GS (transferase) activity and 65-70% in was found in the Av-MHR strain up to 80 mg L-1of all heterocyst frequency to that of untreated control four herbicides tested. The Av-MHR strain exhibited culture in response to Arozin, Butachlor and 2,4-D. 65-72% inhibition in chlorophyll a (Table 1), 50-55% B The Av(M) mutant showed 50% inhibition in in O2 evolution (Table 2), 65-70% in nitrogenase chlorophyll a, 35-69% in photosynthesis, 57-60% in activity (Table 3), 42-46% in GS activity (Table 4) nitrogenase and 20% in GS (transferase) activity to at 100 ppm of all the four herbicides tested. that of untreated control culture in response to Arozin, Similar trend of inhibition was also recorded in Alachlor and 2,4-D. Heterocyst could not be detected macromolecular contents (phycobiliproteins, D in this mutant. The Av(M) showed 48-52% inhibition carbohydrate, DNA, RNA) and dark O2 uptake in chlorophyll a, 39-52% in photosynthesis, 56-62% (data not shown). in nitrogenase, 18-19% in GS (transferase) activity The percent survival curve of Av-MHR isolate and 60-65% in heterocyst frequency as compared to exhibited 70% survival in the combined lethal untreated control culture in response to Arozin, concentrations (25 mg L-1) of all four herbicides as 82 INDIAN J BIOTECHNOL, JANUARY 2012

-1 -1 Table 3—Impact of graded concentration of herbicides on N2-fixation (n mol C2H4 µg chl a h ) of wild type and herbicide resistant mutant strains of A. variabilis

Cyanobacterial Herbicide concentrations strains (mg L-1) 0.0 10.0 15.0 25.0 50.0 85 100

Arozin Av (P) 0.4±0.05 0.2±0.04 0.02±0.01 ND ND ND ND Av (M)Ar-MHR 3.4±0.1 3.4±0.4 3.2±0.4 3.2±0.5 3.0±0.2 2.1±0.2 1.1±0.5 Av (M)Al 1.6±1.0 1.2±0.6 0.6±0.5 0.4±0.1 ND ND ND Av (M)B 0.4±0.2 0.3±0.05 0.2±0.03 0.1±0.03 ND ND ND Av (M)D 1.6±1.2 1.3±1.1 1.1±0.6 0.4±0.06 ND ND ND

Alachlor Av (P) 0.4±0.05 0.3±0.02 0.1±0.02 0.03±0.01 ND ND ND Av (M)Ar-MHR 3.4±0.1 3.4±0.5 3.2±0.7 3.1±0.7 2.2±1.2 1.3±0.8 1.2±0.04 Av (M)Al 1.6±1.0 1.6±1.2 1.5±0.6 1.5±1.0 0.5±0.8 0.4±0.1 0.3±0.1 Av (M)B 0.4±0.2 0.3±0.1 0.2±0.05 0.1±0.05 ND ND ND Av (M)D 1.6±1.2 1.4±1.1 0.7±0.6 0.5±0.04 ND ND ND

Butachlor Av (P) 0.4±0.05 0.3±0.1 0.1±0.05 0.03±0.01 ND ND ND Av (M)Ar-MHR 3.4±0.1 3.4±0.5 3.3±1.0 3.3±0.3 2.9±0.6 1.4±0.8 1.2±0.03 Av (M)Al 1.6±1.0 1.5±0.5 0.6±0.6 0.5±0.03 ND ND ND Av (M)B 0.4±0.2 0.5±0.02 0.5±0.04 0.5±0.02 0.2±0.03 0.1±0.02 0.04±0.01 Av (M)D 1.6±1.2 1.5±1.0 0.6±0.6 0.4±0.2 ND ND ND

2,4-D Av (P) 0.4±0.05 0.2±0.03 0.1±0.01 0.02±0.01 ND ND ND Av (M)Ar-MHR 3.4±0.1 3.4±1.0 3.3±0.6 3.3±0.4 2.3±0.2 1.2±0.1 1.0±0.04 Av (M)Al 1.6±1.0 1.4±1.0 0.5±0.3 0.4±0.03 ND ND ND Av (M)B 0.4±0.2 0.2±0.03 0.2±0.02 0.1±0.01 ND ND ND Av (M)D 1.6±1.2 1.6±0.6 1.6±0.7 1.5±0.5 0.5±0.03 0.4±0.05 0.2±0.03

The data are expressed as mean±SE ND: Not detectable

compared to wild type (data not shown). The it could be a single mutational event. This also combined effect of lethal concentration of all four suggests the presence of herbicide (Arozin, Alachlor, herbicides in wild type strain showed 95, 84, 70, 64, Butachlor, 2,4-D) resistant genes on single loci on the 60, 52, 44, 43, 42, 38 and 37% inhibition in genomic DNA. Possibly a change in the gene nitrogenase activity, GS activity, photosynthesis, responding to alkyl group is responsible for single phycobilins, chlorophyll a, O2 uptake, heterocyst locus mutation, as all the four herbicides are carrying frequency, protein, DNA, RNA, carotenoid and alkyl groups and thus their site of attachment in the carbohydrate, respectively after 24 h of exposure. cyanobacterial system might have been altered as a Almost similar magnitude of inhibition was observed result of mutation. Similarly five herbicide resistant in Butachlor, Alachlor and 2,4-D resistant mutants phenotypes to Machete (Butachlor), Basalin, DCMU, after 24, 72 and 96 h of incubation r (data not shown); Atrazine and Propanil have been reported to be whereas no such reduction was observed in the genetically linked in Gloeocapsa sp.28. Av-MHR isolate under similar conditions (Table 5). It is evident from the results that, in increasing concentration of herbicides, the parent strain showed Discussion substantial retardation in growth and metabolic In the present study, the cross resistance activities, whereas no such decline was recorded in relationship in Arozin resistant mutant strain of the Av-MHR strain. This suggests that the multiple A. variabilis with other herbicides (Alachlor, herbicide resistant mutant has been accompanied by Butachlor, and 2,4-D) has indicated that the resistance acquisition of tolerance in growth and vital metabolic to Arozin is associated with the acquisition of activities against all the four herbicide tested. The resistance to Alachlor, Butachlor and 2,4-D; therefore, resistance that is manifested by the ability of MHR SINGH et al: ISOLATION AND CHARACTERIZATION OF A MULTIPLE HERBICIDE RESISTANT STRAIN 83

Table 4—Impact of graded concentration of herbicides on GS activity (nmol γ-glutamylhydroxamate mg-1 protein min-1) of wild type and herbicide resistant mutant strains of A. variabilis

Cyanobacterial Herbicide concentrations strains (mg L-1) 0.0 10.0 15.0 25.0 50.0 85 100

Arozin Av (P) 3192±55 2617±56 2499±59 480±42 ND ND ND Av (M)Ar-MHR 2645±66 2640±35 2640±28 2640±49 2634±35 1920±66 1424±68 Av (M)Al 2655±50 2432±52 2120±39 523±36 ND ND ND Av (M)B 2650±60 2230±38 2114±52 520±38 ND ND ND Av (M)D 2645±45 2340±48 2140±61 532±62 ND ND ND

Alachlor Av (P) 3192±55 2756±52 2525±65 580±40 ND ND ND Av (M)Ar-MHR 2645±66 2645±62 2642±39 2642±65 2638±62 2130±63 1544±69 Av (M)Al 2655±50 2650±45 2649±41 2648±36 1245±35 986±46 840±39 Av (M)B 2650±60 2228±61 2118±44 526±47 ND ND ND Av (M)D 2645±45 2346±52 2147±43 530±39 ND ND ND

Butachlor Av (P) 3192±55 2700±53 2100±63 540±41 ND ND ND Av (M)Ar-MHR 2645±66 2644±50 2640±66 2640±47 2640±50 2114±70 1534±68 Av (M)Al 2655±50 2534±62 2234±48 534±38 ND ND ND Av (M)B 2650±60 2650±36 2642±41 2640±56 1220±51 980±39 852±33 Av (M)D 2645±45 2350±54 2154±45 532±47 ND ND ND

2,4-D Av (P) 3192±55 2721±58 2520±60 430±43 ND ND ND Av (M)Ar-MHR 2645±66 2640±39 2639±57 2639±67 2636±39 1942±55 1420±56 Av (M)Al 2655±50 2430±47 2118±58 524±32 ND ND ND Av (M)B 2650±60 2232±55 2110±56 510±40 ND ND ND Av (M)D 2645±45 2643±43 2642±44 2640±52 1254±51 975±36 836±35

The data are expressed as mean±SE ND: Not detectable

Table 5—Combined effect of lethal dosages of Arozin, Alachlor, Butachlor and 2,4-D on macromolecular contents -1 -1 -1 (µg mL ), heterocyst frequency (%), photosynthetic O2 evolution, dark O2 uptake (µmol O2 mg chl a min ), nitrogenase activity -1 -1 -1 -1 (nmol C2H4 µg chl a h ) and GS activity (nmol γ-glutamylhydroxamate mg protein min ) of wild type and multiple herbicide resistant mutant strain of A. variabilis

Parameters A.variabilis A. variabilis (wild type) (MHR)Ar,Al,B,D

Chlorophyll a (0.25±3.2) 0.10±1.5 (0.23±2.3) 0.44±2.2 Carotenoid (0.65±2.3) 0.40±2.3 (0.60±2.6) 0.65±2.4 Phycocyanin (2.40±2.4) 0.85±2.3 (3.2±2.4) 3.8±2.3 Phycoerythrin (1.11±2.0) 0.40±3.1 (2.0±2.5) 2.7±1.0 Protein (15.0±2.6) 8.5±3.6 (18.7±0.9) 19.3±1.5 Carbohydrate (20.2±0.8) 12.8±3.8 (20.7±0.5) 21.2±3.5 DNA (8.7±0.8) 5.0±0.8 (8.9±1.2) 9.2±1.6 RNA (14.8±2.1) 8.6±1.6 (15.0±0.6) 15.8±1.7 Heterocyst frequency (4.5±2.0) 2.5±1.2 (4.2±3.6) 4.2±2.2 Photosynthesis (22.0±1.9) 6.5±2.2 (23.0±3.2) 23.5±2.3 O2 uptake (2.5±1.9) 1.2±2.5 (2.5±0.8) 2.5±2.8 Nitrogenase activity (0.4±1.6) 0.02±2.6 (3.7±1.2) 3.7±2.7 GS activity (2850±0.5) 450±2.6 (2645±1.0) 2645±1.6

The data are expressed as mean±SE; The values in parenthesis represent the initial value (0-h) ND: Not detectable Data recorded after 24 h of incubation

84 INDIAN J BIOTECHNOL, JANUARY 2012

cells to grow in the presence of lethal concentration of treated with growth toxic dosages of common rice herbicides may originate from several possible field herbicides. changes in the organism caused by the mutation29. The mutant might have developed an exclusion Acknowledgement mechanism for the herbicide or exhibit detoxification Authors are thankful to the Head, Department of by enhanced degradation. In addition, a modification Biological Science, R D University, Jabalpur (M.P.), of the target enzyme (or protein) might be the basic India, for facilities and the Department of Science and mechanism of resistance. This possibility includes Technology, Government of India, New Delhi, for over production of the target enzyme or alteration in financial assistance. its amino acid sequence leading to reduced efficiency of herbicide binding30-32. A comparison of given References physiological and biochemical characteristics of the 1 Singh R N, Role of blue green algae in nitrogen economy of Indian agriculture (Indian Council of Agricultural Research, parent strain under herbicide stress suggest that all New Delhi) 1961, 17-19. herbicides adversely affected N2-fixation when tested 2 Roger P A, Biological N2-fixation and its management in individually (Table 3) or applied together (Table 5). wetland rice cultivation, Fertil Res, 42 (1995) 261-276. Alachlor, Butachlor and 2,4-D resistant mutants also 3 Rai A N & Bergman B, Creation of new nitrogen fixing exhibited marked sensitivity in nitrogenase activity cyanobacterial associations, Biol Environ: Proc R Irish Acad, 102B (2002) 65-68. upon exposure to the herbicides tested. 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