Marc LEGRAS 1 Christophe GANGNEUX 1 Pierre PLASSART 1 Daniel TESSIER 3 Jeanne-Chantal DUR 3 Caroline BAILLEUL 1 Salima TAIBI 2 Karine LAVAL1
Effect of physico-chemical characteristics of agricultural soils on fungal biomass August 20-25, 2006, Vienna Austria
1 Laboratoire BioSol
The main membrane sterol of most fungi is the ergosterol. It is generally used for estimating the living fungal biomass in high plants, stored crops, feedstuffs or plant litter. For the soils, the fungal biomass is now usually determined by PFLA patterns, but this requires expensive equipment like mass spectrometry. The fungal biomass is used as a biomarker to assess the soil fertility and to supervise the effects of an environmental pollution.
2 LAMSAD École d’Ingénieurs en Agriculture Esitpa – 13, rue du Nord 76000 Rouen - France
INTRODUCTION
1
Our aim is to be able to use the ergosterol as an indicator of change of the agricultural practice. We tried to optimize certain techniques of extraction, while basing ourselves on usual methods and to emphasize the effect of various variable on this marker.
3 Unité PESSAC (Physico-Chimie et Ecotoxicologie de SolS d'Agrosystèmes Contaminés) INRA Versailles-Grignon Batiment de Science du Sol Route de St-Cyr 78026 Versailles Cedex - France
Table 1: Physico-chemical characteristics of the plots
Study sites Field plots on silty soils were located in north-western France (Normandie). There are 2 experimental sites (1 and 2) including each one a permanent meadow (PM) and a traditional crop (TC) where the pedoclimatic conditions supported the development of an intensive agriculture. The climate is mild with a mean rainfall of 800 to 900 mm/y and low seasonal ranges. The field plots characteristics are summarized in the table 1.
%
%
g/kg
g/kg
dry weight basis
g/kg
dry weight basis
dry weight basis
Sampling The samples were collected in April, June, August and October 2005. 20 composite samples were collected from the layers 0-10 cm of each field plot. All samples, kept at 4°C until analyse, were sieved (2 mm) in the field moist condition. All values are expressed on a dry weight basis (105°C, 24 h). Cmol+/kg
%
bulk density
dry weight basis
Analytical determination (HPLC-DAD) of fungal biomass (ergosterol)
2
Total ergosterol: Micro waves assisted Extraction - MAE For extraction, 1.2 g fresh soil was put into 50mL high pressure reactor (TFM Quartz®) and mixed with NaOH 2M and MeOH. The extraction treatment was a grouped irradiation in a specific microwave oven (Mars X®) for 2 x 15 s with 15 min intermission. After the treatment, the soils and solvents, of each reactor, are recovered by 2 x 4 mL of MeOH. For the step of purification, we added 3 x 4 mL of pentane, each addition was shaken by vortex for 10 s and centrifuged for 2 min at 2000 RPM. Pentane was precisely taken and evaporated by pure nitrogen flow. The bottles were taken again by 2 mL of MeOH and filtered directly in the HPLC vials before quantification (1), (3).
Ergosterol Extractions in soils As Fig. 1 shows it, the second extraction (MAE) shows results higher than the first (without saponification). The first extraction seems to quantify the free molecules of ergosterol in the soil. They are supposed to come from fungal cell membranes in degradation. The second technique also extracts the molecule present in the form of ester within the viable membranes of the cells. We obtain that result whatever the agricultural practice (meadow or crop) and the periods of sampling (April, June, August, October). Regarding to the bibliography, there are several interpretations on these two types of ergosterol. We adopt the approach that free ergosterol corresponds to the “dead” fungal biomass and that the MAE extraction gives access to the total ergosterol (dead and alive biomass).
µg ergosterol / g dry soil
MATERIAL AND METHODES
Free ergosterol: without saponification Soil (4g of moist soil) was combined with acid-washed glass beads and methanol in a 20-mL scintillation vial. The vial was vortexed for 10 s and then shaken for 1 h at 320 RPM on an orbital shaker. After shaking, the soil-methanol mixture was allowed to precipitate for 15 min and an aliquot of 1,5 mL supernatant was centrifuged for 10 min at 11,000 RPM. The supernatant was then filtered and filled in a HPLC vial. (2)
7,00 6,00
Free ergosterol
5,00
Total ergosterol
4,00 3,00 2,00 1,00 0,00 1 2
3 4 5
6
7
9,00
Crop - TC2
8,00
Meadow - PM2
Crop - TC1 Meadow - PM1
6,00 5,00 4,00 3,00 2,00 1,00 0,00 1 2
RESULTATS AND DISCUSSION
3
µg ergosterol / g dry soil
µg ergosterol / g dry soil
10,00
9,00 7,00
3 4 5
6
7
7,00 6,00 5,00 4,00
Ergosterol versus fungal DNA
3,00 2,00
9
3 4 5
6
11 12 13 14 15 16 17 18 19 20
7
8
9 10 11 12 13 14 15 16 17 18 19 20
soil samples
Fig. 2a: Total ergosterol contents in the PM1 and TC1 plots June experiments
Fig. 2b: Total ergosterol contents in the PM2 and TC2 plots June experiments
Linear Correlations (Pearson test): Free ergosterol vs DNA 18S : 0.72 Total ergosterol vs DNA18S : 0.31
Results show that the mean values of fungal biomass are higher in meadows than in crops (on both sites and for all the experimentation periods). In the same way, their spatial variations are less in crops than in meadows. Fig. 2a (site 1) and Fig. 2b (site 2) illustrate these two points, strong ergosterol contents with a strong variability in the both meadows, and less ergosterol contents in crops with more constant values. On one hand, 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. On the other hand, the crops had been plowed, they were drier, the plant cover and the rhizosphere were under development. The values are thus weak and homogeneous.
Cationic exchange capacity
2506010
2506010
2506010
2506000
2506000
2506000
2506000
mg/kg
17
2505980
2505990
12.5 12.4 12.3 12.2 12.1 12 11.9 11.8 11.7 11.6 11.5 11.4 11.3 11.2 11.1 11 10.9 10.8 10.7 10.6 10.5 10.4 10.3
2505980
16 15
2505970
2505970
14 13
2505960
12
2505960
11 2505950
10
2505950
9 2505940
8
2505940
7 2505930
6 4
2505910
2
2505900
2505910
517440
517460
517480
517500
517520
517420
517440
517460
517480
517500
2506000
2505970
2505960
2505930
2505920
2505910
2505960
10.5 10 2505950
9.5 9
2505950
2505940
8.5
2505940
8 7.5
2505930
7 6.5
517500
6
2505920
2505910
5.5 5
2505910
2505900
517440
517460
517480
517500
2505960
2505950
2505940
2505930
2505920
2505910
517460
517480
517500
517520
517460
517480
517500
517520
Apparent density 2506000
%o 2505990 340 330 320 310 300 290 280 270 260 250 240 230 220 210 200 190 180 170 160 150
2505980
2505970
2505960
2505950
2505940
2505930
2505920
2505910
2505900
517440
517440
2506010
2505990
2505970
517420
517420
517520
2506000
70 68 66 64 62 60 58 56 54 52 50 48 46 44 42 40 38 36 34 32 30 28 26 24
2505980
517520
2505930
2505920
g/kg 2505990
2505900
2505900
517480
2505960
Clay
2506000
34 33.5 33 32.5 32 31.5 31 30.5 30 29.5 29 28.5 28 27.5 27 26.5 26 25.5 25 24.5 24 23.5 23 22.5 22 21.5 21 20.5
2505980
517460
2505970
11.5 11
2506010
% 2505990
8.2 8 7.8 7.6 7.4 7.2 7 6.8 6.6 6.4 6.2 6 5.8 5.6 5.4 5.2 5 4.8 4.6 4.4
2505980
12
517420
517520
2506010
517440
13 12.5
2505970
Organic Matter
Humidity 2506010
517420
2505990 14 13.5
2505980
2505900
2505900
2505940
Fig. 3: Space distribution of various variables measured on a plot ( PM1 - April experiments)
2505920
3
2505950
4
2505930
5 2505920
517420
Cmol+/kg
g/kg 2505990
18
There is no direct correlation point to point in a plot between a physico-chemical variable and fungal biomass. Nevertheless, it seems that cation exchange capacity (r=0,53) and the pH (r=0,56) play a significant role in the contents of ergosterol. Moreover, by taking into account all the data of the study, there is a good relation between the apparent density and the total ergosterol content (r=0,88).
pH
2506010
2505990
Physico-chemical characteristics of agricultural soils
Non-Linear Correlations (Spearman test): Free ergosterol vs DNA 18S : 0.68 Total ergosterol vs DNA18S : 0.69
C/N
Total ergosterol
CONCLUSION REFERENCES
There is good correlations point to point between free ergosterol and the fungal biomass measured by real time PCR (fungal DNA). Results show a good representativeness of ergosterol for the fungal biomass and the functional difference between free and total ergosterol.
1,00 0,00 1 2
8
soil samples10
9 10 11 12 13 14 15 16 17 18 19 20 les
Fig. 1: Ergosterol contents in the PM1 plot – June experiments
10,00 8,00
8
meadow sa mp
Effect of the agronomic practice on the fungal biomass
517420
1.5 1.48 1.46 1.44 1.42 1.4 1.38 1.36 1.34 1.32 1.3 1.28 1.26 1.24 1.22 1.2 1.18 1.16 1.14 1.12 1.1 1.08 1.06 1.04 1.02 1 0.98 0.96
2505980
2505970
2505960
2505950
2505940
2505930
2505920
2505910
2505900
517440
517460
517480
517500
517520
517420
517440
517460
517480
517500
517520
Both extraction methods give access to two types of fungal biomass (degraded or total biomass). The mean values of fungal biomass are higher in meadows than in crops and their spatial variations are less in crops than in meadows. As for the ergosterol contents, the mean values of the physico-chemical characteristics behave in the same way (higher contents in meadows, variations are less in crops). But there is no direct correlation between the physico-chemical characteristics of the soil and the fungal biomass (at the plot scale). →A multiparametric modeling is necessary to reach a better understanding of the effect of the various variables. →Ergosterol, as a marker of the fungal biomass, is eligible to be an indicator of the soil quality. (1) Mongomery et al. (2000) Determination of soil fungal biomass from soil ergosterol analyses. Soil Bio Biochem 32 : 1207-1217. (2) Gong et al. (2001) A rapid method to extract ergosterol from soil by physical disruption. Appl Soil Ecology 17 : 285-289. (3) 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.
*
[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.
