Biodegradation of the fungicide metalaxyl by zygomycetes

Abstract

A large amount of fungicides, such as metalaxyl [methyl N-(methoxyacetyl)-N-(2,6-xylyl)-DL-alaninate], are currently used against Oomycetes species that cause downy mildew in several agricultural crops, including vineyards. However, these compounds are potentially harmful for terrestrial and aquatic environments, as well as to human health due to their carcinogenic and mutagenic properties. Recently, there has been an increase in interest in using filamentous fungi, such as Zygomycetes, which degrade xenobiotic compounds and other recalcitrant molecules using nonspecific extracellular enzymes, for the bioremediation of pesticide in polluted soils. In this study, several Zygomycetes strains, Gongronella spp., Absidia spp., Circinella spp. and Rhizopus spp., were used to screening their degradation metalaxyl ability, using solid and liquid cultures. In order to identify and characterise these taxa a polyphasic approach including morphology characterization, molecular fingerprint M13-PCR and Matrix Assisted Laser Desorption Ionization Time of Flight Intact Cell Mass Spectrometry (MALDI-TOF ICMS) were used. After selective enrichment on solid medium containing a fungicide gradient concentration raging of 0-100 mg.L-1, strains Absidia glauca CBS 101.08 and Rhizopus orizae CCMI 900, showed capacity to tolerate high metalaxyl concentrations. These resistant strains were selected to perform liquid assays using Yeast Nitrogen liquid cultures supplemented with sucrose (5 g.L-1) and metalaxyl (100 mg.L-1). Biomass concentration was determined by dry weight. A. glauca CBS 101.08 and R. orizae CCMI 900 showed specific growth rates of 0.774 h-1 and 0.999 h-1, respectively. Sucrose was totally consumed up to 5 days of culture, with a sucrose consumption rate of 0.93 and 0.84 g.L-1. day-1 for A. glauca CBS 101.08 and R. orizae CCMI 900, respectively. The non-degraded metalaxyl in liquid cultures was determined by UV-HPLC and evaluated periodically, during 21 days. The metalaxyl degradation rate for A. glauca CBS 101.08 and R. orizae CCMI 900 was 2.22 mg.L-1.day-1 and 2.29 mg.L-1.day-1, respectively. Results suggest that A. glauca CBS 101.08 and R. orizae CCMI 900 strains can be explored in soil bioremediation for metalaxyl degradation. These strains are now under study to determine the presence of extracellular enzymes that could be involved in the metalaxyl degradation process.