Effect of metal speciation on the functional and morphological responses of soil fungi: case of Cu and Zn in Trametes versicolor

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Effect of metal speciation on the functional and

morphological responses of soil fungi: case of Cu and Zn in Trametes versicolor

Jérémie Lebrun, Albert Kollmann, Nelly Wolff, Isabelle Lamy, Christian Mougin

To cite this version:

Jérémie Lebrun, Albert Kollmann, Nelly Wolff, Isabelle Lamy, Christian Mougin. Effect of metal

speciation on the functional and morphological responses of soil fungi: case of Cu and Zn in Tram-

etes versicolor. 5. ISMOM International Symposium of Interactions of Soil Minerals with Organic

Components and Microorganisms Commmission, Nov 2008, Pucon, Chile. 1 p, 2008. �hal-02824546�

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1 Influence of Metal Speciation on the Sensitivity of Functional Influence of Metal Speciation on the Sensitivity of Functional and Morphological Responses to Cu and Zn in

and Morphological Responses to Cu and Zn in Trametes versicolor Trametes versicolor

J

Jé ér ré émie mie Lebrun Lebrun, Albert , Albert Kollmann Kollmann, Nelly Wolff, Isabelle , Nelly Wolff, Isabelle Lamy Lamy and Christian and Christian Mougin Mougin

INRA

INRA --UR251: PESSAC UR251: PESSAC --PhysicochemistryPhysicochemistryand and EcotoxicologyEcotoxicologyof of SoilSSoilSfrom Contaminated from Contaminated AgrosystemsAgrosystems, Versailles, FRANCE, Versailles, FRANCE

1- 1 - INTRODUCTION INTRODUCTION

Filamentous fungi are key actors of soil functioning because of their ability to produce extracellular enzymes. Soil contamination by metals can alter these extracellular activities as well

as fungal morphology. The impact will depend on metal bioavailability, which is linked to metal speciation. But the way the metal speciation influences the sensitivity of fungal responses is not well known, in particular in the case of essential metals Cu or Zn. Knowledge of metal speciation and its relationships with biological traits are important for the development of tools for metal ecotoxicity assessment, and could enhance the predictive interest in such fungal biomarkers.

4 4- - RESULTS RESULTS

5 5- - CONCLUSION CONCLUSION

Free forms of metals: Free forms of metals: LesaLesa< Aba5 < Aba2.5< Aba5 < Aba2.5

5

^th^th

ISMOM, ISMOM, Pucó Puc ón n (Chile), Nov. 2008 (Chile), Nov. 2008

2- 2 - OBJECTIVE OBJECTIVE

The present study aims at establishing the influence of metal speciation on both specificity and sensitivity of fungal responses to essential metals.

For that purpose, Trametes versicolor was cultured in liquid media exhibiting different levels of metal complexation. Three functional and two physiological traits were compared for their

pertinence as metal exposure biomarkers.

3- 3 - METHODS METHODS

I. CULTURE MEDIA and METAL SPECIATION

- Use of three liquid media performed from Kirk medium (pH 5.5) differing only by their content in organic ligands :

- Metal speciation was determined theoretically using a thermodynamic program (SOILCHEM) and validated experimentally using a Cu²⁺-specific electrode - Addition of copper in the different media yielded a good

relationship: experimental vs theoretical [Cu²⁺] values ; R²> 0.96

II. FUNGAL RESPONSES

- Cultures of Trametes versicolor in the different media - Addition of Cu or Zn from 1 nM to 1 mM the 3^thday of culture - Exposure during one week to essential metals

A. PHYSIOLOGICAL RESPONSES

- Determination of dry biomass as a toxicity/tolerance indicator - Observations by scanning electron microscopy to characterize

morphological impacts

B. FUNCTIONAL RESPONSES

- Measurements of 3 extracellular oxidase activities in liquid media:

lignin-peroxidase ; Mn-peroxidase ; laccase Liquid medium Complexing

properties Organic

ligands LesaLesa Aba5Aba5 Aba2.5Aba2.5 2.5 g/l 5 g/l

1 g/l Potentially Yeast extracts

- -

50 mM Highly

Tartric acids

*

**

*

* *

**

0 2000 4000 6000 8000

Ctrl 7 6 5 4 3

- Log (added Cu)

Laccase activity (U/g d.w.)

*

**

*

**

0 100 200 300 400

Ctrl 7 6 5 4 3

Mn-peroxidase activity (U/g d.w.)

**

*

* *

**

** **

0 50 100 150

Ctrl 7 6 5 4 3

Lignin peroxidase activity (U/g d.w.)

I. CULTURE MEDIA and METAL SPECIATION II.A. PHYSIOLOGICAL RESPONSES (Growth and Morphology)

II.B. FUNCTIONAL RESPONSES - Metals are mainly complexed by organic ligands in Lesa (>80%)

-The presence of free metal species are favored in Aba5 and Aba2.5 (until 13% for Cu²⁺and 74% for Zn²⁺, in red Fig. 1)

- Zn has no effect on the oxidase activities, whatever the complexation level of the used medium

- Laccase and Mn-peroxidase activities are highly increased by Cu in low complexing media Aba5 and Aba2.5

Fig. 3:□^Control■Zn at 1 mM ■Cu at 1 mM ; values ± SEM (n=9 ; *p<0.05)

The The intensityintensityof enzymatic responses is increasedof enzymatic responses is increased when free forms of Cu are favored; it is not the case for Zn when free forms of Cu are favored; it is not the case for Zn

Zn Zn²²⁺⁺

Cu Cu²⁺²⁺

organic organic and inorganic and inorganic elements elements METAL SPECIATION METAL SPECIATION

METAL METAL EXPOSURE EXPOSURE

FUNGAL FUNGAL RESPONSES RESPONSES

-

-Extracellular oxidases:Extracellular oxidases:

•

•Lignin-Lignin-peroxidaseperoxidase

••MnMn--peroxidaseperoxidase

•

•LaccaseLaccase --Growth (dry biomass)Growth (dry biomass) -

-Morphology of myceliumMorphology of mycelium At morphological level At morphological level

At functional level At functional level

- The growth of T.versicolor is not significantly affected by the essential metals whatever the medium, except with Cu at 1 mM and in Aba2.5 (data not shown)

- The fungal morphology is not affected by Cu while Zn seems to induce a coalescence of mycelia but only at 1 mM and in low complexing media Aba5 and Aba2.5 (Fig. 2)

Fig. 2: Scanning electron microscopy of T.versicolor mycelia cultured in the medium, Aba2.5 (Magnification: x 2000) Cu

Cu ZnZn

Control (1 mM) (1mM)

Despite some physiological alterations, Despite some physiological alterations, T. versicolorT. versicoloris tolerant to Cu and Znis tolerant to Cu and Zn

Fig. 4: Culture of T. versicolor in Aba5 □and Aba2.5 ■; values ± SEM (n=9 ; *p<0.05)

- The threshold appearance of oxidase stimulation is lowered in the media Aba5 and Aba2.5 from 1 mM (in Lesa, Fig. 3) to 1µM (Fig. 4)

- Lignin peroxidase is specifically expressed in the presence of Cu only in these low complexing media

The The sensitivitysensitivityof enzymatic responses to Cu is increasedof enzymatic responses to Cu is increased when the metal is more bioavailable

when the metal is more bioavailable a. Oxidase responses to essential metals (1 mM) b. Dose-response curves to Cu in low complexing media

*

0 * 2000 4000 6000 8000

Les Aba5 Aba2.5

Medium

Laccase activity (U/g d.w.)

*

0 100 200 300 400

Les Aba5 Aba2.5

Medium

Mn-peroxidase activity (U/g d.w.)

Cu

Cu CuCu CuCu

0 20 40 60 80 100

Lesa Aba5 Aba2.5

Medium

Metal forms of Cu (%)

Cu Cu

0 20 40 60 80 100

Lesa Aba5 Aba2.5

Medium

Metal forms of Zn (%)

Fig.1: ■organic-complexed, ■inorganic-complexed and □^{free forms} Zn

Zn

74 % 13 % 28 %

5 %13 % 1 %

[email protected] J. Lebrun is grateful to INRA and the region Haute-Normandie for their financial support The response of Trametes versicolor is specific to one essential metal and

depends on metal speciation. We found that Zn leads to some morphological alterations at high concentration levels while Cu highly stimulates extracellular oxidase activities at environmental levels of metal contamination.

These enzymatic actors of soil functioning can be biomarkers of fungal exposure to Cu in soils. Furthermore, the specificity and sensitivity of fungal responses to metals confirm that fungal traits can be used as tools for metal ecotoxicity assessment.

Studies are now engaged to improve a quantitative extraction of enzymes from soils.