World Journal of Microbiology and Biotechnology, 8, 326-328. Short Communication. Effect of pesticides on growth, respiration and nitrogenase activity of ...
World Journal of Microbiology and Biotechnology, 8, 326-328
Short Communication
Effect of pesticides on growth, respiration and nitrogenase activity of Azotobacter and Azospirillum S.A. Omar and M.H. Abd-Alla* Pesticides (Brominal, Cuprosan and Fenvalerate) at 10 and 50 ppm suppressed growth, respiration and nitrogenase activity of Azotobacter chroococcum, AzospiriIIum brasilense and Azospirillum ]ipos The inhibitory effect on respiration of Asm. Iipoferum was most pronounced after 3 and 4 days. Key words: Azospirillum, Azotobacter, fungicide, herbicide, insecticide, nitrogenase activity, respiration
Azotobacter and Azospirillum are non-symbiotic N2-fixing bacteria and are common and important members of the rhizosphere microflora (Hegazi et al. 1979). In agriculture, chemical agents are often used to control crop losses caused by microorganisms and insects, as well as unwanted grasses growing amongst the crops. One of the biggest ecotoxicological problems in Egyptian agriculture is the intensive use of pesticides which can attack non-target organisms, including soil microorganisms. Under the United States of America Toxic Substances Control Act, one of the proposed microbiological assays requires microorganisms involved in nitrogen-cycling to be tested for their sensitivity to potential toxins (Stem 1980). Among various microbial activities in soil, Nz-fixation by diazotrophs contributes significantly to the overall nitrogen economy of soils. The effect of several pesticides on N2-fixation and nitrogen cycle was extensively studied in the soil by Nayak & Rao (1982). Reports on the effect of pesticides on growth, respiration and Nz-fixation in pure culture are, however, scarce. The present work has investigated the effect of three pesticides on the physiology of three diazotrophic bacteria, chosen because of their ecological and agricultural importance, in a laboratory environment. S.A. Omar and M.H. Abd-Alla are with the Department of Botany, Faculty of Science, University of Assiut, Assiut, Egypt. ~ Corresponding author.
9 1992Rapid Communications of Oxford Ltd
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Materials and Methods N2-Fixing Bacteria Azotobacter chroococcum was obtained from the Unit of Bio-Fertilizers, Department of Microbiology, Faculty of Agriculture, Ain Shams University, Egypt. Azospirillum lipoferum 215 and Azospirillum brasilense245 were provided by Dr J. D6bereiner (Empresa, Brosileira de Pesquisa, Agropecuaria, 23460 Seropedica, Rio de Janeiro, Brazil).
Bacterial Culture Conditions Abr. chroowccum was grown in Brown's nitrogen-flee liquid medium (Brown et al. 1962). The Azospirillum spp. were grown in semi-solid N-free medium (D6bereiner 1978). From a 48-h culture of each organism (grown on nitrogen-flee media), 0.2 ml were inoculated into 100-ml Erlenmeyer flasks, each containing 30 ml of nitrogen-flee medium. Pesticide Testing One fungicide (Cuprosan), one herbicide (Brominal) and one insecticide (Fenvalerate) were examined. Each was sterilized in distilled water and added to sterilized media to give 0, 10 and 50 ppm. Cultures were incubated at 30~ without shaking. Growth was evaluated by protein content using Lowry's method, nitrogenase activity by the acetylene reduction method (Hardy et al. 1968) and the amount of CO2 evolved by control and treated cultures by the method of Cheng & Coleman (1989).
Pesticides inhibit asymbiotic N2-fixation
Results and Discussion
chroococcum after 4 days growth in medium with 10 ppm of
There was a progressive decrease in the growth of all organisms and of nitrogenase activity with increasing concentrations of all three pesticides (Table 1). Cuprosan (a mixture of two thiocarbamates and copper oxychloride fungicides) was less toxic to the three organisms than Brominal (a benzoic acid herbicide) or Fenvalerate (a pyrethroid insecticide). The nitrogenase activity of Abr.
Table 1. Effect of pesticides on protein content end nltrogenase activity of Azotobacter chroococcum (a), Azospirillum brasilense 245 (b), and Azospirillum Ilpoferum 215 (c). All values were determined 72 h alter treatment. Pesticides (ppm)
Control 0 Brominal 10 50 Cuprosan 10 50 Fenvalerate 10 50
Protein content (/~g ml-1 culture)
Nitrogenase activity (nmol ethylene ml-1 culture h -1)
a
b
c
a
b
c
245
343
240
271
48
34
225 101
134 97
70 45
177 88
25 15
24 10
240 120
151 102
125 102
186 97
31 28
27 11
180 101
124 84
50 35
140 86
12 10
9 9
Each value represents the mean of three replicates. All experimental values were significantly different from control values.
these pesticides appeared more resistant to their toxic effects than that of Asm. lipoferum 215 or Asm. brasilense 245. Bacterial respiration (Table 2) varied according to pesticide concentration and the organism itself. COz evolution by Abr. chroococcum and Asm. brasilense 245 was significantly lowered by all pesticides under all conditions. With Asm. lipoferum 215, however, respiration was significantly promoted by most treatments after 24 and 48 h but was decreased after 72 or 90 h. The toxic effect of herbicides such as 2,3,6-trichlorobenzoic acid and pentachlorophenol to Azotobacter has been reported (Tam & Trevors 1981; Ferrer et al. I986). The toxic effect of the pyrethroid insecticide, Fenvalerate, to Abr. chroococcum, Asm. brasilense and Asm. lipoferum could be due to its effect on nitrogenase, as the degree of growth inhibition was similar to that of ethylene production in the acetylene-reducing assay. This finding agrees with Mano et al. (1988), who also observed that growth and nitrogenase activity of Asm. lipoferum were inhibited in nitrogen-free media amended with the organochloride insecticide Dicofol above I ppm. The low nitrogenase activity in the presence of pesticides was accompanied by an inhibitory effect on growth and respiration. However, Ferrer et al. (1986) reported that 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid and 2,3,6-trichlorobenzoic acid did not affect microbial respiration. Tam & Trevors (1981) showed that the low acetylene reduction activity in the presence of pentachlorophenol could possibly be due to its inhibitory effect on oxidative phosphorylation and ATPase activity, since a correlation between Oz uptake and electron transport activity in some organisms had been established.
Table 2. Effect of pesticides on respiration of Azotobacter chroococcum (a), Azospirillum brasilense 245 (b) and Azospirillum Iipoferum 215 (c), expressed as pg C02/30 ml growth culture/24 h. Pesticides (ppm)
Days alter treatment 1
2
3
4
a
b
c
a
b
c
a
b
c
a
b
c
0 Brominal
31
17
27
93
26
35
75
12
36
81
11
30
10 50 Cuprosan
11" 16"
16" 18
57* 32*
54* 31"
18" 12"
37* 33*
51" 45*
11" 9*
44* 36
21" 44*
8* 6*
20* 17"
10 50 Fenvalerate
4* 25*
9* 14"
52* 38*
88* 56*
27* 30*
52* 63*
73* 35*
13" 3*
36 30*
90* 98*
17" 45*
35* 12"
10 50
11" 12"
12" 11"
16" 27
52* 83*
27* 17"
50* 28*
52* 41"
16" 3*
23* 30*
36* 32*
23* 6*
9* 3*
Control
* Significant difference from the control. Each value represents the average of three replicates.
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S.A. Omar and M.H. Abd-Alla Studies (Barnes et al. 1973) have shown that some pesticides are degraded in moist soil. However, the decomposition under field conditions is relatively slow and pesticides may persist in soil for long periods, suggesting that frequent applications of Brominal, Fenvalerate and Cuprosan in agricultural soils may result in accumulation of high amounts of these compounds with consequent inhibition of microbial nitrogenase activity. Extrapolation of the effects observed in culture solutions to field condition demands caution. Laboratory studies give only an idea of the possible effect. Therefore, further studies under field conditions are necessary.
References Barnes, R.D., Bull, A.T. & Poller, R.C. 1973 Studies on the persistence of the organotin fungicide, Fentin acetate (Triphenyltin acetate) in the soil and on surfaces exposed to light. Pesticides Science 4, 305-317. Brown, M.E., Burlingham, S.K. & Jackson, R.M. 1902 Studies on Azotobacter species in soil. I. Comparison of media and techniques for counting Azotobacter in soil. Plant and Soil 17, 320-332. Cheng, W. & Coleman, D.C. 1989 A simple method for measuring CO z in a continuous air-flow system: Modification to substrate-induced respiration technique. Soil Biology and Biochemistry 21, 385-388.
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D6bereiner, J. 1978 Influence of environmental factors on the occurrence of Azospirillum lipoferum in soil and roots. Environmental role of N2-fixing blue-green algae and asymbiotic bacteria. Ecological Bulletin (Stockholm) 26, 343-352. Ferrer, M.R., Gonzalez-Lopez, J. & Ramos-Cormenzana, A. 1980 Effect of some herbicides on the biological activity of Azotobacter vinelandii. Soil Biology and Biochemistry 18, 237-238. Hardy, R.W.F., Holsten, R.D., Jackson, E.K. & Bums, R.C. 1968 The acetylene-ethylene assay for nitrogen fixation. Laboratory and field evaluation. Plant Physiology 43, 1185-1207. Hegazi, N.A., Monib, M. & Vlassak, K. 1979 Effect of inoculation with Nz-fixing spirilla and Azotobacter on nitrogenase activity on roots of maize grown under subtropical conditions. Applied and Environmental Microbiology 38, 621-625. Mano, D.M.S., Matos, A.C.M. & Langenbach, T. 1988 The effect of Dicofol on morphology, growth and nitrogenase activity of Azospirillum lipoferum. In Azospirillum IV: Genetics, Physiology, Ecology, ed Klingmuller, W. pp. 159-165. Berlin and Heidelberg: Springer-Ver[ag. Nayak, D.N. & Rao, V.R. 1982 Pesticides and nitrogen fixation in a paddy soft. Soil Biology and Biochemistry 14, 207-210. Stem, A.M. 1980 Role of microorganisms in environmental assessments. In Microbiology 1980, ed Schlessinger, D. pp. 361-365. Washington: American Society for Microbiology. Tam, T.Y. & Trevors, J.T. 1981 Toxicity of pentachlorophenol to
Azobacter vinelandii. Bulletin of Environmental Contamination and Toxicology 27, 230--234. (Received in revised form 6 January 1992; accepted 14 January 1992)
