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