Effect of physico-chemical characteristics of agricultural soils on fungal biomass (FB) – Impact of copper pollution 2
Marc LEGRAS 1, Caroline BAILLEUL 1, Daniel TESSIER2, Jeanne-Chantal DUR 2, Christophe GANGNEUX 1 , Salima TAÏBI3, Karine LAVAL1
1 Laboratoire BioSol 3 LAMSAD École d’Ingénieurs en Agriculture Esitpa – 13, rue du Nord 76000 Rouen - France
INTRODUCTION
Agricultural soils, and specifically cropped soils, constitute ecosystems highly disturbed by human activities. The agricultural management of soils has a great impact upon the functional process of soil microbial communities, particularly on fungal biomass (FB). In that context, our objective was to define the equilibrium status of the soil in order to estimate how pollutants can modify this reference status. For that purpose, we measured the variability of 40 physicochemical and biological quantitative descriptors in soils from cropped and meadow plots during three years, using field experiments and microcosms of undisturbed soils (for contaminated soils). This study focuses on fungal community and the impact of copper contamination on this microbial compartment.
Unité PESSAC (Physicochimie et Ecotoxicologie de SolS d'Agrosystèmes Contaminés) INRA Versailles-Grignon Bâtiment de Science du Sol Route de St-Cyr 78026 Versailles Cedex - France
MATERIAL AND METHODES Study sites
Field plots on silty soils are located in north-western France (Yvetot - Normandie). There are a permanent meadow (M) and a traditional crop (C) where the pedoclimatic conditions supported the development of an intensive agriculture. The climate is mild with an average rainfall of 800 to 900 mm/y and low seasonal ranges. The field plots characteristics are summarized in the Table 1.
Sampling
Fields sampling have been realized in order to observe temporal impact. The samples were collected in April, June, August and October from the layers 0-10 cm of each field plot. Each descriptors was measured at each date for both soils (200 samples). We developed microcosms (Fig. 1) fitted with columns of undisturbed soil from the previous fields to assess the ecotoxicity of chemical pollutants and carried out the experimentations for 2 months (90 samples, 3 dates). These microcosms were settled in the ground under external conditions (climate, temperature, moisture and pressure). Setup of microcosm experiments: 45 columns of each agricultural soil (15 references, 15 inoculated with Cu to obtain 2ppm content, 15 inoculated with Cu to obtain 200 ppm content). 5 samples of each situation were analyzed after 7, 21 and 60 days. All samples (fields and microcosms), were kept at 4°C one night until analyze, were sieved (2 mm) in the field moist condition. All values are expressed on a dry weight basis (105°C, 24 h).
Figure 1: Microscosm setup – Inoculation of copper at T0 – Before analysis
Determination of fungal biomass: 5 descriptors
The FB has been measured by using several approaches in this work: (i) genetic using real time PCR (18S rDNA) (Gangneux et al. 2004) (ii) chemicals, by extraction and quantification of total ergosterol (Legras et al. 2004), free ergosterol (Gong et al. 2001) and specific PLFAs (C18:2w6,9, C16:1w5). 18S rDNA targets conservated sequences of DNA in the fungi kingdom. Free ergosterol has been extracted without saponification and seems to quantify the free molecules of ergosterol in soil. They are supposed to come from fungal cell membranes in degradation. Total ergosterol is obtained by Micro waves assisted Extraction and with saponification. This descriptor take into account the ergosterol molecules present in the form of ester within the viable membranes of the cells. Specific PLFAs are extracted by SPE columns and resulting FAMEs are analyzed by GC-FID. C16:1 is specific of fungi in soils, C18:2 more specific of the arbuscular mycorrhizal fungi (Baath et al. 2003). 2 The descriptors: fungal DNA, ergosterol, PLFAs
RESULTS AND DISCUSSION
Whatever the approach considered (18S rDNA, ergosterol, PLFAs) very significant correlations (Table 2) have been found indicating that molecular and chemicals protocols are relevant to access FB. Moreover, the results show strong rank correlations (ergosterol vs C16:1, ergosterol vs 18S rDNA) and linear correlation (ergosterol vs C18).
Physico-chemical data of the experimental plots Table 1. Means of physico-chemical characteristics of the soils from Yvetot. Permanent meadow and intensive crop.
Table 2. Correlation coefficients ( r ) and p-value. (a) linear correlations (Pearson), (b) rank correlations (Spearman).
Effect of the agronomic practices on the fungal biomass
8,00
Crop
8,00
Meadow
7,00
Crop
6,00
Meadow
18S rDNA (ppm dry soil)
Total ergosterol (ppm dry soil)
9,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 0,00 1
2
3
4
5
6
7
8
soil samples
FAME of C16:1 (ppb dry soil)
9,00
10,00
5,00 4,00 3,00 2,00 1,00 0,00
9 10 11 12 13 14 15 16 17 18 19 20
Figure 3a: Total ergosterol contents in the M and C plots June experiments
1 2 3 4 5 6 7 8 soil samples 9 10 11 12 13 14 15 16 17 18 19 20
4,00 3,50
Crop
3,00
Meadow
2,50 2,00 1,50 1,00 0,50 0,00
Figure 3b: 18S rDNA contents in the M and C plots June experiments
1 2 3 4 5 6 7 8 9 soil samples 10 11 12 13 14 15 16 17 18 19 20
Figure 3c: FAME of C16:1w5 contents in the M and C plots June experiments
The analysis of all fields data shows that all the descriptors of fungal compartment are relevant to discriminate two contrasted agricultural soils. Fig. 3 indicates that the fungal biomass in the meadow is higher than in the crop whatever the descriptor (same results for free ergosterol and C18:2) throughout the experiments. In the same way, their spatial variations are less in crops than in meadows (strong fungal contents with a strong variability in the meadow, and less fungal contents in crops with more constant values). At one point, this difference can be explained by having at the same time, the presence of grass, a moisture and a rhizosphere more significant in the meadows. Moreover, the crops had been plowed, they were drier, the plant cover and the rhizosphere were under development.
Impact of copper on the fungal biomass
Relations between physico-chemical characteristics and the fungal biomass
Table 3. Linear correlation coefficients and p-value.
T= 7 days
Meadow
8
Meadow
4
T= 60 days
3,5
7
Fungal contents (ppm dry soil)
There is strong correlations, dot per dot in a plot, between physicochemical variables and FB. It seems that C, N, and the pH play a significant part in the contents of FB. Nevertheless, the temporal evolution of fungal community in the microcosm compared to the copper impact revealed that its impact is not significant (Fig. 4).
T= 7 days T= 60 days
4,5
9
6
3
5
2,5
4
2
3
1,5
2
1
1
0,5 0
0 2
18S rDNA
Total ergosterol
1,8
Crop
1,8
Crop
1,6
1,6
1,4
1,4
1,2
1,2
1 1
0,8
0,8
CONCLUSIONS The ANalysis Of Variance of the whole data enable us to classify the different factors affecting FB: - Agricultural practices constitute the main factor, - The sampling date (season, soil management, climate…) is the second one, - Total nitrogen and carbon, apparent density and pH are major variables in the behavior of the FB, - we were unable to evidence any significant effect of copper on the micro-organisms of the soil, especially for FB, using that unusual setup of microcosms
0,6
0,6
0,4
0,4
0,2
0,2
0
0
Field reference
without Cu
with 2ppm of Cu
with 200ppm of Cu
Field reference
without Cu
with 2ppm of Cu
with 200ppm of Cu
Figure 4 Contents of fungal biomass, (1)18S rDNA and (2) total ergosterol, in the field at T=0 day and microcosms (without, with 2ppm or 200ppm of copper) for the both agricultural practices at T=7days and T=60days.
REFERENCES Baath et al. (2004) Can the extent of degradation of soil fungal mycelium during soil incubation be used to estimate ecomycorrhizal biomass in soil? Soil Biology & Biochemistry 36:2105-2109. Gangneux et al. (2004) Real-time PCR to investigate telluric fungi, Eurosoil 2004, Freiburg, Germany, 4th-12th September 2004. Gong et al. (2001) A rapid method to extract ergosterol from soil by physical disruption. Appl Soil Ecology 17 : 285-289. Legras et al. (2004) A fast and sensitive method to determine the fungal biomass in the agricultural soils, Eurosoil 2004, Freiburg, Germany, 4th-12th September 2004.
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[email protected] - http://www.esitpa.org/biosol Acknowledgements: The authors thank the ADEME Support (grant n° 0475C0071) the LGTA from Yvetot and the commune of Saint-Georges-sur-fontaine for the access to the various sites.
