Background . Laccases, mostly laccase-like multicopper oxidases (LMCO), are probably the most common ligninolytic enzymes in soil. Although, in recent studies, laccase-encoding genes have been successfully used as molecular markers in order to elucidate the role of bacteria in soil organic C cycling , further research in this field is necessary . In this study, using rainfed olive farming as an experimental model, we determined the stability and accumulation levels of humic substances and appliedthese data to bacterial laccase-encoding gene expression and diversity in soils under four different agricultural management systems (bare soils under tillage/no tillage and vegetation cover under chemical/mechanical management).
Materials and Methods. Humic C (>10 4 Da) was subjected to isoelectric focusing. The GC-MS method was used to analyze aromatic hydrocarbons. Real-Time PCR quantification and denaturing gradient gel electrophoresis ( DGGE) of DNA/RNA for functional bacterial laccase-like multicopper oxidase (LMCO)-encoding genes and transcripts were also carried out.
Results. Soils under spontaneous vegetation, eliminated in springtime using mechanical methods, showed the highest humic acid levels as well as the largest bacterial population, rich in laccase genes and transcripts after more than 30 years of experiments. The structure of the bacterial community based on LMCO genes also pointed to phylogenetic differences between these soils due to the impact of different management systems. Soils where herbicides were used to eliminate spontaneous vegetation once a year and those where pre-emergence herbicides resulted in bare soils clustered together for DNA-based DGGEanalysis, which indicated a certain amount of microbial selection due to the application of herbicides. When LMCO-encoding gene expression was studied, soils where cover vegetation was managed either with herbicides or with mechanical methods showed less than 10% similarity, suggesting that the different laccase substrates derived from vegetation cover decay when herbicides are used.
Conclusions. We suggest that the low humic acid content retrieved in the herbicide-treated soils was mainly related to the type (due to vegetal cover specialization ) and smaller quantity (due to lower vegetal biomass levels) of phenolic substrates for laccase enzymes involved in humification processes. We also found that spontaneous vegetal cover managed using mechanical methods could be the best option for achieving C stabilization in rainfed Mediterranean agroecosystems.
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