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Materials and methods

The structure of the microbial population was studied using two molecular biology methods for analysing the DNA of soils (PCR-TTGE and PCR-T-RFLP).

Soils samplings

Soil sampling was carried out on the GWS and MSW treatment plots, as well as on the control treatments receiving or not receiving complementary nitrogen fertilisation. This sampling was carried out on seven dates, from September 2004 to October 2006, or just after the fourth spreading (2004) and one month after the fifth spreading (2006). These dates are shown in Table 1:

Table 1. Dates of soil sampling for the analysis of microbial communities.

Prélèvements

P1

P2

P3

P4

P5

P6

P7

P8

P9

Dates

09/2004

11/2004

04/2005

06/2005

10/2005

09/2006

10/2006

Nombre de mois après le dernier épandage

24*

2

7

9

13

24*

1

* Sample S1 is taken just after the spreading of 2004. Sample S6 is taken just after the spreading of 2005.

The microbial communities were analysed by comparing the samples taken from the treatments on the same date. This is because different dates produce different results due to seasonal cycles. For each treatment, monitoring can be carried out over time in order to observe time-based changes in microbial populations.

Analysis of microbial communities

Two molecular methods were used to analyse the DNA of soils (PCR-TTGE and PCR-T-RFLP). These two methods produce qualitative data on the microbial diversity of the soil. The bacterial and fungal sections were studied. The principle is to extract and purify the DNA contained in the soil.
This DNA is amplified by PCR. Then, in the case of PCR-TTGE, the DNA is fragmented using a restriction enzyme (Figure 1). Analysing these fragments then enables the microbial populations to be identified. In the case of PCR-T-RFLP, the DNA is amplified with primers that amplify characteristic DNA sequences that allow the microbial populations to be identified (Figure 2).

T-RFLP_72dpi

Figure 1. Diagrammatical representation of the T-RFLP (Terminal Restriction Fragment Length Polymorphism) analysis of DNA sequences amplified by PCR using a primer marked by fluorochrome and digested by a restriction enzyme.

TTGE_72dpi

Figure 2. Diagrammatical representation of the TTGE (Temporal Temperature Gradient gel Electrophoresis) analysis of DNA fragments amplified by PCR using a "clamped" primer and separated by electrophoresis using a temperature gradient.