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Influence of Xenobiotic Substances on Actinomycete Comunities in Soil

Marioara Nicoleta Filimon1, Roxana Popescu2, Aurica Breica Borozan3, Despina Maria

Bordean4, Gabi Dumitrescu5, Sorin Octavian Voia5

1West University of Timioara, Faculty of Chemistry, Biology and Geography, Department of Biology-Chemistry, 300115-Timioara, Pestalozzi, 16, Romania

2University of Medicine and Pharmacy Victor Babes, Faculty of Medicine, Department of Cellular and Molecular Biology, 30004-Timioara, E. Murgu, 2, Romania

3Banats University of Agricultural Sciences and Veterinary Medicine from Timisoara, Faculty of Horticulture and Sylviculture, 300645-Timioara, Calea Aradului, 119, Romnia

4Banats University of Agricultural Sciences and Veterinary Medicine from Timisoara, Faculty of Food Processing Technology, 300645-Timioara, Calea Aradului, 119, Romania

5Banats University of Agricultural Sciences and Veterinary Medicine from Timisoara, Faculty of Animal Science and Biotechnologies, 300645-Timioara, Calea Aradului, 119, Romania

Abstract Sulfonylurea herbicides are frequently used in agricultural crops even if they determine quantitative and qualitative changes in soil microbial communities. In this study it was used increasing doses of two sulfonylurea herbicides, tribenuron-methyl and nicosulfuron, in order to establish their effect on actinomyces communities from soil under laboratory conditions. Using nutritive gelose with soil extract and Gause medium the main species of actinomyces were identified: Streptomyces albus, Streptomyces aureus and Streptomyces chrysomallus. Streptomyces albus is the most abundant species, which could indicate a resistance to used herbicides. Sulfonylurea herbicides, tribenuron-methyl and nicosulfuron, point out a lower value, inhibiting the actinomycete communities from soil.

Keywords: actinomycetes, sulfonylureic herbicid, xenobiotic 1. Introduction Herbicides are highly important for agriculture. Sometimes, these herbicides may act as pollutants, damaging the soil, ground water and surface water. Bacterial communities have a significant importance in assuring herbicide degradation. Actinomyces play an important role in cycle of the elements in nature and in degradation of organic xenobiotic substances [1], and also is an important group in production of antibiotics and metabolism byproducts [2].

*Corresponding author: Marioara Nicoleta Filimon, Tel.: +40256592646, Fax: +40256592622, Email: nicoleta_filimon@yahoo.com

Sulfonylureic herbicide degradationv (chlorsulfuron and imazosulfuron) may be chemically or biologically done. Biological degradation may be achieved with great efficiency with the help of bacteria communities in soil. These kind of bacteria can easily survive in alkaline soil treated with herbicides [3]. The growth of actinomyces in soil is strongly influenced by the type of vegetation, content of total nitrogen, total phosphorus, total potassium and available potassium. Significant correlations were found between actinomyces communities, streptomyces and the parameters said before [4]. The effects of certain herbicides like quizlofop-P-tefuril (Pantera), oxasulphuron (Dynam), flumioksazin (Sumisoya) + quizlofop-P-tefuril (Pantera) and flumioksazin (Sumisoya) +

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oxasulphuron (Dynam) were studied to establish the effects on the dynamics of soil biogeny. The following parameters were analyzed: total number of microorganism, abundance of Azotobacter, fungi, and actinomyces and the dehydrogenase activity was also studied. The abundance of fungal and actinomyces communities increased while the abundance of Azotobacter significantly decreased. This indicates that these two groups of microorganisms use the herbicides as a source of biogenic elements [5]. Other papers studied the effect of four herbicides (atrazine, primextra, paraquate and glyphosate) on bacterial, actinomycete and fungi communities from soil samples which were incubated for six weeks under laboratory conditions. All studied communities showed a decrease compared to control sample [6]. But other studies reports increase in fungi and actinomycete communities after glyphosate treatment [7], or show no significant changes on long term in microbial populations [8]. The aim of this study is to establish the effect of two tribenuron-methyl and nicosulfuron herbicides on actinomycete communities from soil in laboratory conditions. 2. Materials and methods Soil samples were collected from a cambic chernozem at depth ranges of 0-20 cm, from the experimental field of Plant Breeding discipline, the U.S.A.M.V.B. department in Timioara. The collected soil samples have been brought to the laboratory and treated with two herbicides. The herbicides were represented by tribenuron-methyl and nicosulfuron. Treatments of soil samples were performed under laboratory conditions. Tribenuron-methyl known as 2-methox-6 -[[[[( 4-methyl-1 ,3,5-triazine-2-il) methylamino] carbon] sulfone] benzoic amino acid is also present in herbicides from local markets under the trade name Helmstar (Tellurium Chemical, Romania). Nicosulfuron known as 2 - [[(4,6-dimethoxypyrimidin-2-il) aminocarbon] aminosulfon]-N, N-dimethyl-3-pyridinecarboxamide is also present in herbicides from local markets under the trade name Mistral (ISK Biosciences Europe, Romania).

Soil treatment with herbicides The soil was sifted through a sieve of 2 mm and placed in polyethylene bags. Different doses of herbicides have been prepared with distilled water and applied to the ground as a part of moisture in order to ensure 40% soil moisture. Herbicide application on dry soil was calculated assuming a uniform distribution of herbicides in the plow layer [9]. An untreated sample was preserved, while experimental samples were performed with increasing doses of herbicide. The following experimental variants were obtained after applying the herbicides: group-normal doses (ND) (A1-0.6 g tribenuron-methyl, A2-nicosulfuron 0.4 g), group B-2xND (B1-1.2 g tribenuron-methyl, B2-nicosulfuron 0.8 g), group C-3xND (C1-1.8 g tribenuron-methyl, C2-nicosulfuron 1.2 g), group D-5xND (D1-3 g tribenuron-methyl, D2-nicosulfuron 2 g), group E-7xND (E1-4.2 g tribenuron-methyl, E2-nicosulfuron 2.8 g). The herbicides were applied separately in the above mentioned doses. Samples were incubated for 7 days in a thermostat at 28 C. Subsequently quantitative and qualitative analyses of fungal colonies were performed. Isolating and identifying the actinomycetes Actinomycetes isolation has been established as it follows: dilutions have been made from soil samples using the suspension method decimal dilutions; the 10-6 dilution of every experiment has been inoculated on an environment with soil extract and an additional meat broth [10]. The inoculation was made with the help of an inoculum dissemination technique on the environment surface. Subsequently, the incubation was carried out in a thermostat with the plates turned upside down, at a temperature of 28 C, during a period of 5-7 days. The actinomycetes communities have been afterwards moved to a Gause culture environment [11], incubated in a thermostat, at a temperature of 28 C, during a period of 7 days. Streptomyces sp. have been notified with the help of Gause`s method. Statistical data interpretation Statistical data interpretation has been performed using Past Statistical Program version 2.12 Freeware.

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3. Results and discussion The main species of Streptomyces genus found in the experimental variants were: Streptomyces aureus, Streptomyces albus and Streptomyces chrysomallus (Figure 1, 2, 3).The discussion should interpret the results clearly and concisely.

Figure 1. Streptomyces albus

Figure 2. Streptomyces aureus

Figure 3. Streptomyces chrysomallus

Using the PCA statistical analysis, we established that the Streptomyces aureus species is representative to the C1 experimental variant (soil treated with tribenuron-methyl 3xND). Streptomyces albus is well represented by A2, D2 and control experiments. The Streptomyces chrysomallus species may be found at the D2 experiment (soil treated with cu nicosulfuron 5xND) (Figure 4).

Figure 4. PCA Actinomycetes experimental variants Streptomyces albus is the most abundant species from every experiment. It is also the most resistant to xenobiotic substances (Figure 5).

Figure 5. PCA Actinomycetes communities

Streptomyces albus is one of the most dominant species according to the diversity index and Shannon`s method, indicating a value of 1,099 (Figure 6).

Figure 6. PCA of experimental variants

Streptomyces aureus may be also found in the control experiment. In this case, there are no important changes, as the herbicide doses are higher. Streptomyces albus is not present in the control experiment, but the species are found in certain experiments, when nicosulfuron herbicide is added. This is due to the fact that these species are resistant to xenobiotics.

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Studies concerning the effect of other sulfonylureic herbicides (metsulphuron-methyl) on actinomycete communities in soil reveal that their number has been reduced. It states that these species are sensitive to xenobiotic substances [12]. Severe inhibition effects of actinomycete communities have been also recorded by Pampulha et al. [13]. Soil samples have been treated with glyphosate-ammonium herbicides in laboratory conditions, during 40 days. Based on higher doses of herbicide, it has been established that the Streptomyces aureus is significantly reduced (Figure 7).

Figure 7. PCA Studied experimental variants

4. Conclusions Streptomyces albus is one of the most important species from the experimental variants, indicating that these species are resistant to xenobiotic actions. Reducing actinomycetes communities from soil treated with 2 types of herbicides indicate the fact that these species are sensitive to the action of xenobiotic substances. Sulfonylureic, tribenuron-methyl and nicosulfuron herbicides point out a lower value, inhibiting the actinomycetes community in soil. References 1. McCarthy, A.J., Lignocellulose-degrading actinomycetes. FEMS Microbial. Rev., 1987, 46, pp. 145163.

2. Strohl, W.R., Antimicrobials. In: Microbial diversity and bioprospecting (Bull, A.T., Ed.), American Society for Microbiology, Washington, DC, 2004, pp.336355. 3. Wang, YS., Chen, WC., Lin, LC., Yen, JH., Dissipation of herbicides chlorsulfuron and imazosulfuron in the soil and the effects on the soil bacterial community. Journal Environ Sci Health Part B., 2010, 45, pp. 449-455. 4. Yang, B., Xue, QH., Chen, ZQ., Guo, ZY., Zhang, XL., Zhou, YQ., Xu, YJ., Sun, DF., Ecological distribution and antimicrobial effects of soil actinomycetes in artificial vegetation systems in Shazhuyu of Qinghai, China, 2008, 19(8), pp. 1694-701. 5. Cvijanovi, G., Miloevi, N., Cvijanovi, D., Priji, L., The Dynamics of Soil Biogeny Parameters After the Apllication of Herbicides, 3rd European Conference on Pesticides and Related Organic Micropollutans in the Evironment, Halkidiki, Greece, 2004, pp. 335-338. 6. Sebiomo, B., Ogundero, VW., Bankole, SA., Effect of four herbicides on microbial population, soil organic matter and dehydrogenase activity. African Journal of Biotechnology, 2011, 10(5), pp. 770-778. 7. Araujo, ASF., Moniteiro, RTR., Abarkeli, RB., Effect of glyphosate on the Microbial activity of two brazillian soils. Chemosphere, 2003, 52, pp. 799- 804. 8. Busse, MD., Ratcliffe, AW., Shestak, CJ., Powers, RF., Glyphosate toxicity and the effects of long term control on soil microbial communities. Soil Biol. Biochem., 2001, 33, pp. 1777-1789. 9. Atlas, RM., Parmer, D., Partha, R., Assessment of pesticide effects on non-target soil microorganisms. Soil Biol. and Biochem., 1978, 10, pp. 231-239. 10. Zarnea, Gh., Degradarea microbian a substanelor xenobiotice, Tratat de microbiologie general. Ed. Academiei Romane. 1994, pp. 976-991. 11. Gause, GF., Vopros Klasifikatii Actinomitetov-Antagonistov, Medghiz, Moskva, URSS, 1967. 12. He, Y., Shen, D., Fang, C., Zhu, Y., Rapid degradation of metsulfuron-methyl by a soil fungus in pure cultures and soil. World Journal of Microbiology and Biotechnology, 2006, 22 (10), pp. 1095-1104. 13. Pampulha, ME., Ferreira, MASS., Oliveira, A., Effects of a phosphinothricin based herbicide on selected groups of soil microorganisms. Journal of Basic Microbiology, 2007, 47(4), pp. 325331.