Biodegradation Of Metalaxyl And Its Effects On Soil Microbial Community With Repeated Applications

Pesticide is an important component of modern agriculture, and contributes a great deal to pest control and improvment of the yield and quality of crops. However, the widely used pesticides would have adverse effects on non-target microorganisms in soils. Metalaxyl(methyl-N-(2,6-dimethyl)-N-methoxyacetyl-D,L-alaninate) is an important acylanilide fungicide developed by Ciba-Geigy Co.Ltd., characterized by low toxicity and long residual period, and is widely used to control plant diseases of downy mildew, late blight, damping off and stem and fruit rots of many plants caused by Oomycetes, Phycomycetes and other fungi in several crops by inhibiting fungal protein synthesis in oomycete. Metalaxyl is soluble in water(8.4 g/L, 22℃), and available in several commercial formulations. It has the stability of hydrolysis, photolysis in water and soil and does not volatilize slightly and has strong resistance over a wide range of pH and temperature. On account of its physiochemical properties and broad-spectrum systemic activity, metalaxyl is used on a broad range of crops such as vegetables, fruits and horticultural crops etc. Metalaxyl has been registered for use worldwide including in Asia, Europe, USA, Australia, India and Egypt. Metalaxyl is used as a seed treatment for banded or broadcast soil application and as a foliar spray. However, metalaxyl with a single application to soil can disturb the microbial community. The above results were obtained under the condition of single application of metalaxyl. The consequences of continuous inputs of fungicide to soil may differ from those of a single application. In fact, repeated applications of metalaxyl are necessary to control the fungal diseases in field. However, studies focusing on the variation of degradation rates and effects on soil microbial community with repeated applications of metalaxyl are limited. Therefore, it is essential to investigate the degradation rate of metalaxyl and the influence of the fungicide on soil microbial community with repeated applications to soil. In this study, laboratory and field experiment were conducted to examine the dissipation rates along with the effects of successive applications of metalaxyl at two different rates on the soil microbial community through characterizing the soil microbial community structure and diversity(DGGE). The results are as following:A laboratory experiment was conducted to study the impact of metalaxyl repeated application for 2 and 3 times at different concentration levels of Recommended Dose and Double the Recommended Dose on microbial community(DGGE and T-RFLP) and the degradation of metalaxyl in soil. The degradation of metalaxyl followed a pseudo-first-order kinetic reaction and degradation rates of metalaxyl were dependent on the application frequency. It is remarkable that dissipation of metalaxyl was accelerated with its increasing application frequency. This indicated that repeated applications of metalaxyl resulted in the growth of microorganisms capable of degrading metalaxyl. The same variation in soil bacteria and actinomycetes population was observed in treatments with two successive inputs of metalaxyl to soil compared with the control. Soil bacteria and actinomycete were inhibited significantly during the first 14 d of incubation and stimulated on the following 42 days of experiment compared to the control. For fungi, the soil microbial number decreased significantly on day 28 and then recovered to the level of the control at the end of the experiment after 2 repeated applications of metalaxyl. However, no obvious change in soil bacteria population was observed on day 7 but followed by significant increase on day 14 and 28, then, the number of bacteria decreased to the level of the control. The successive applications of metalaxyl inhibited significantly the growth of the fungi. For actinomycete, microbial number increased compared to the control after the third treatments. The above results suggested that metalaxyl was toxic to soil fungi but bacteria and actinomycete can use metalaxyl as carbon resource and energy to grow. Results of DGGE and T-RFLP indicated that soil bacterial community structure experienced an obvious alteration after 2 and 3 repeated metalaxyl applications, some sensitive bacteria were killed, specific populations were enriched, but some other gene bands did not alter throughout the experiment, indicating developed tolerance to metalaxyl under selective pressure. Repeated applications of metalaxyl significantly increased diversity of bacteria throughout the experiment. Bacillus subtilis subsp. Spizizenii strain 168 played an important role in metalaxyl degradation.A field experiment was conducted to investigate the degradation rate and the effects on soil microbial communities of repeated 2 and 3 times of applicated metalaxyl. The degradation of metalaxyl followed a pseudo-first-order kinetic reaction and degradation rates of metalaxyl were dependent on the application frequency, and dissipation of metalaxyl was accelerated with its increasing application frequency. The degradation rates of metalaxyl in field were higher than in laboratory, resulting from the influence of temperature or rainfall. Soil bacterial and actinomycetic population were increased, and fungal population were inhibited. The above results indicated that metalaxyl was slightly toxic to soil fungi, but could be used as resources of carbon and enegy to growth. Results of DGGE and T-RFLP indicated that some sensitive bacteria were killed, specific populations were enriched, but some other gene bands did not alter throughout the experiment, indicating developed tolerance to metalaxyl under selective pressure with repeated metalaxyl applications, so repeated applications of metalaxyl significantly altered diversity and insturcture of bacterial community throughout the experiment.A laboratory experiment was conducted to study the impact of metalaxyl repeated application for 2 and 3 times at different concentration levels of Recommended Dose and Double the Recommended Dose on microbial community(DGGE and T-RFLP) and the degradation of metalaxyl in soil incubated phytophthora parasitica var. nicotianae. The degradation rates of metalaxyl in soil inoculated phytophthora parasitica var. nicotianae were the same as those without phytophthora parasitica var. nicotianae. This indicated that there were no obvious competition between phytophthora parasitica var. nicotianae and indigenous microorganisms with 2 repeated application of metalaxyl. However, phytophthora parasitica var. nicotianae propagated and competed with the microorganisms able to degrade metalaxyl, resulting in induced population of the microbes. Soil bacterial population increased, and fungal population were inhibited, actinomycetic population were inhibited on the first 14 d with 2 repeated applications of metalaxyl, but were stimulated on 28 d of 2 repeated applications and after 3 repeated applications of metalaxyl. The above results were similar to those in soils without phytophthora parasitica var. nicotianae, except for the bacterial population on the first 14 d after 2 repeated applications of metalaxyl. Results of DGGE and T-RFLP indicated that some sensitive bacteria were killed, specific populations were enriched, but some other gene bands did not alter throughout the experiment, indicating developed tolerance to metalaxyl under selective pressure with repeated metalaxyl applications, so repeated applications of metalaxyl significantly altered diversity and insturcture of bacterial community throughout the experiment.