J. Basic Microbiol. 40 (2000) 4, 279 – 288 ( 1 Photobiological Nitrogen Fixation Research Laboratory, Department of Genetics and Plant Breed- ing, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi-221005 India and 2 Institut für Botanik und Pharmazeutische Biologie, Friedrich-Alexander-Universität, Staudtstr. 5, D-91058 Erlangen, Germany) A cyanobacterial mutant resistant against a bleaching herbicide A. VAISHAMPAYAN 1 , R. P. SINHA 1, 2 , A. K. GUPTA 1 and D.-P. HÄDER 2 (Received 25 January 2000/Accepted 17 April 2000) The nitrogen fixing cyanobacterium Nostoc muscorum has been found to be sensitive to the herbicide SAN 6706 [4-chloro-5 (dimethylamino)-2-(a,a,a,-trifluoro-m-tolyl)-3-(2H) pyridazinon] at 30–45 μM within 15 min. The toxicity was more severe in combined nitrogen-free (N comb -free) medium than in a combined nitrogen medium; this enhancement was reversible by supplementation of the medium with 3 mM glucose or 5 μM ATP, serving as carbon and/or energy source in this organism. A mutant of this cyanobacterium resistant to 3 mM SAN 6706 has been isolated and characterized to perform nitroge- nase activity in exogenous ATP supplemented N comb -free medium. However, it exhibited a moderate growth in combined nitrogen media in the absence of external ATP. The resistance factor is higher than 100. Simultaneously, this strain possesses a cross-resistance to methylamine, a well-known inhibitor of photophosphorylation, irrespective of the exogenous ATP supply. The behavior of the mutant suggests a defective phosphorylation in its photosynthetic system. Application of herbicides in agriculture is a wide spread practice to suppress weed growth and increase crop yields (CHATTOPADHYAY 1987). However, massive use of these chemi- cals poses a potential danger to agriculturally important N 2 -fixing cyanobacteria (GREAVE 1982, ZARGAR and DAR 1990, VAISHAMPAYAN et al. 1998). Most of the earlier studies on this aspect have been confined to screening for the lethal and semi-lethal concentrations of various agro-chemicals in cyanobacteria with little emphasis on the possible mode(s) of action in these organisms (VENKATARAMAN and RAJYALAKSHMI 1972, MEHTA and HAWXBY 1979, ROGER and KULASOORIYA 1980). The present work attempts to improve the situation, reporting results on the biological effects of the herbicide SAN 6706 [4-chloro-5 (dimethylamino)-2-(a,a,a,-trifluoro-m-tolyl)- 3-(2H) pyridazinon] on the N 2 -fixing cyanobacterium Nostoc muscorum by isolation and physiological characterization of a unique mutant resistant to 3 mM SAN 6706 showing an absolute dependence on exogenous ATP for N 2 fixation and growth in N comb -free me- dium. Materials and methods The organism: The filamentous, heterocystous and N 2 -fixing cyanobacterium Nostoc muscorum, isolated from a paddy field in Darbhanga, India (axenic strain No. Nm 01 of the L. N. M. University Culture Collection Centre, Darbhanga, India), was cultured in modified Chu 10 medium (GERLOFF et al. 1950) supplemented with 5 mM KNO 3 . The organism neither forms heterocysts nor fixes nitro- gen in the presence of external nitrogen, such as NO 3 – , NO 2 – or NH 4 + , but on transfer from a combined N medium to a fresh N comb -free medium it starts to form heterocysts (with a frequency of 5–6 per hun- dred vegetative cells) and fixing nitrogen within 20–24 h. Like other wild type isolates of Nostoc