ECOFUN-MICROBIODIV: an FP7 European project for developing ... 11-50.pdf · pesticides impact on the on soil microbial communities and do not reveal consequences on soil ecosystem

ECOFUN-MICROBIODIV: an FP7 European project for developing and evaluating innovative tools for assessing the impact of pesticides toxicity on soil

microbial diversity and functions

Martin-Laurent F.1, Petrić I.2, Kandeler E. 3, Djuric S.4 and Karpouzas D.5

5 research teams:

1 Institut National de la Recherche Agronomique, Dijon, France

2 Institut Rudjer Boskovic, Zagreb, Croatia

3 University of Hohenheim, Stuttgart, Germany

4 Faculty of Agriculture, Novi-Sad, Serbia

5 University of Thessaly, Larissa, Greece

Pesticide Behaviour in Soils, Water and Air, 2-4 September 2013, York, UK

http://www.uns.ac.rs/en/prozor.htm

https://www.uni-hohenheim.de/startseite.html?&L=1

http://www.uth.gr/en

http://www4.inra.fr/ecofun_microbiodiv


ECOFUN-MICROBIODIV: an FP7 European project for developing and evaluating innovative tools for assessing the impact of pesticides toxicity on soil

microbial diversity and functions

http://fabricemarti8.wix.com/ecofun-microbiodiv


Context: the impact of herbicides on soil ecosystem

Despite the pivotal role of microorganisms in ecosystem functioning the assessment of pesticides soil microbial toxicity is lagging behind the recent

methodological advances in soil microbiology


European Soil Framework Directive – identifies agricultural practices as a major threat for soil biodiversity

• Modified Sturm test : estimation of pesticide

biodegradability and of pesticide on the

biodegradation of a reference substrate [OECD 301B]

• Carbon mineralization test [OECD 217]

• Nitrogen mineralization test [OECD 216]

Estimation of the impact of pesticides on

soil microbiota

These global tests do not provide a comprehensive assessment of pesticides impact on the on soil microbial communities and do not reveal

consequences on soil ecosystem services

Context: EU level pesticide regulation

ECOFUN-MICROBIODIV project aimed at Develop and evaluate innovative tools to estimate the impact of low-dose

herbicides sulphonylureas on the function and population dynamics of broad microbial groups and selected microbial taxa


Scheme: ECOFUN-MICROBIODIV organized in 6 WP

WP4 Agronomical monitoring

WP5 Pesticide monitoring

WP 6 Microbial monitoring

WP3 Set up of field and greenhouse experiments

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WP2 Dissemination - Stakeholder Oriented and Policy Implications

o Novi Sad, Serbia, June to October 2011.

o full randomized block pattern (6m x 5m)

o 3 treatments : x1, x2, x5 of the recommended agronomic dose (80 g a.i. ha-1)

o control plots

o 4 replicate plots per treatment

o Tier II toxicity assessment: representating realistic exposure scenario

Set-up of the field experiment


Zea mays variety NS640

Test compound – sulfonylurea herbicide Nicosulfuron

used for the post-emergence control of annual

grass and broad-leaf weeds in maize

application rate 10-1000 times lower than

conventional herbicides 50-100 g of ai /ha

inhibition of the actohydroxyacid synthase (AHAS) → biosynthesis of valine,

leucine and isoleucine (branched chain amino acids)

Set-up of the greenhouse experiment


o Dijon, France, under controlled conditions

o 5 repeats per treatment

o 3 treatments: x10, x100, x1000 of the recommended agronomic dose (80 g a.i. ha-1)

o control pots

o Tier I toxicity assessment – under extreme long-term exposure scheme

o 5 culture cycles

+/- nicosulfuron

4 leaves stage

repeated short culture cycle (5 weeks)

0 40


Scheme





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o Crop yield measurement and weed development (weeds identification using morphological traits and seeds collected to test for

nicosulfuron resistance)

Agronomic monitoring: efficiency of herbicide

developing of the method for monitoring low-dose herbicide in soil

1) extraction (accelerated solvent extraction, ASE*)

2) sample concentration and cleanup (soild phase extraction, SPE**)

3) residue analysis (HPLC*** equipped with a UV diode array detector system; 245 nm)

Pesticide monitoring: persistance of herbicide in soil


*ASE 200; Dionex, Sunnyvale, CA, USA; ** Strata-X, Strata-NH2 *** Varian, Walnut Creek, CA, USA





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Scheme

Testing pertinence of the existing standard methods

- methods aimed at studying abundance (ISO14240:2), diversity (ISO/TS29843-1) and activity (ISO/TS 22939) of the soil microflora

Develop, test and propose new methods for standardization

- methods based on direct soil DNA extraction (ISO 11063) and further PCR

o qPCR analysis assesing dynamics of the most abundant microbial taxa involved in key ecosystem services (NWI 17601*)

o Arbuscular mycorrhizal fungi as bioindicators – changes in their diversity and community structure

Microbail monitoring - methodology

The combined application of different tools will provide a comprehensive assessment of the effects of herbicide on soil microbial communities

Estimating impact of nicosulfuron on:

1) Soil microbial diversity (structure and abundance)

2) Soil microbial function targeting microbial groups participating in important geochemical cycles

3) Herbicide impact on bacterial populations harboring AHAS gene

4) Estimating adaptation of soil microbes on nicosulfuron

Microbail monitoring - methodology


1) A-RISA, qPCR, PLFA, enzyme activities, arbuscular mycorrhiza fungal colonization, DGGE, sequencing

3) A-RISA, qPCR, pyrosequencing

4) Isolation of nicosulfuron-resistant and nicosulfuron-degrading populations, RFLP, sequencing

2) qPCR (amoAa, amoAb), PLFA

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Field exp. Tier II toxicity +/- - - - - - +

Greenhouse exp. Tier I toxicity + + + + + + +

Synthesis of the results

- = non significantly affected; + = significantly affected as compared witth control

Field experiment: Dissipation


Greenhouse: Accumulation

Repeated treatment in greenhouse experiment yielded in a significant decrease of total bacterial community; Gram-negative and Gram-positive bacterial populations


Impact of nicosulfuron on the structure of soil microbial community by means of PLFA

0,0

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Treatment cycle number

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Measurement of a wide range of enzymes involved in C, N and P cycling covering important ecosystemic services

Functional impact of nicosulfuron on microbial community by enzyme activity measurements

all enzymes were negatively affected by repeated exposure to the highest dose rates of nicosulfuron

0,0

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C cycle β-glucosidase β-xylosidase

N cycle N-acetyl-β-glucosaminidase

P cycle phosphatase

* p<0.05, ** p<0.01, *** p<0.001



Impact of nicosulfuron on structure and abundance of bacterial and fungal community by A-RISA and qPCR

* β-Proteobacteria, Acidobacteria, α-Proteobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Verrucomicrobiales, Firmicutes, Gemmatimonadetes, Crenarchea

*

Field exp.

No impact

Impact (x5 dose) Gemmatimonadetes

Fungal community

Structure

Abundance

Impact (x100, x1000)

No impact

Greenhouse exp.

Bacteria community

Structure

Abundance

No impact

Impact (X100, x1000 dose)

Β-Proteo. , Planctomycetes,

No impact

No impact

Actinobacteria, Firmicutes

15,80%

19,95 %

Cycle 4

Β-Proteobacteria

Fungal community * p<0.05, ** p<0.01, *** p<0.001

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High responsiveness of AM fungi to pollutant exposure and their key role on plant diversity and functioning of above ground ecosystems: ideal bio-

indicators for assessing the soil microbial ecotoxicity of pollutants*

Colonization capacity and community structure of Arbuscular mycorrhizal fungi under nicosulfuron exposure

gradual build up of nicosulfuron residues in the greenhouse exp. induced drastic changes in plant growth, establishment of symbiosis and the diversity and community structure of AM fungi

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Cycle 3

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Cycle 4

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o nicosulfuron impaired (x100 dose) or entirely halted maize growth (x1000 dose) from cycle 2 onwards - mycorrhizal colonization showing similar response

Cycle 1 Cycle 3 Cycle 4

* Establishment of ISO10832 ‘Effects of pollutants on mycorrhizal fungi’

DGGE fingerprinting analysis of the intraradical mycorrhizal community in maize roots

o structure of the AM fungal community was clearly affected by nicosulfuron: higher dose - more significant changes

o clear reduction of the community diversity for x100 dose from cycle 2

o maize roots were mostly colonized by different members of Glomus group

o some members showed tolerance or even a positive response to nicosulfuron while others were clearly sensitive to nicosulfuron

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o resistant mutants = harboring AHAS gene non-sensitive to nicosulfuron

difference in resistant community structure under low and high pressure


Phylogenetic diversity of the NS-resistant populations developed under different exposure schemes

Greenhouse exp.

Field exp.

Bacillus & Arthrobacter - most abundant populations

Specific isolates: Flexibacteraceae, Chitinophaga & Rhizobium

Some genera showed more tolerance to higher doses of nicosulfuron:

Bacillus* - most abundant population (65% of the isolates)

Appearance of specific nicosulfuron resistant sub-populations :

Sphingobium, Burkholderia, Cupriavidus & Sphingobacterium


Proposing new method to International Standard Organisation

The combined application of well-standardized tools of different resolution level can provide a comprehensive assessment of the effects of pesticides on soil

microbial functions and population dynamics

Standardization = a 6 steps process

Acceptance of the new work item (ISO 17601) ‘Estimation of abundance of selected microbial gene sequences by qPCR from DNA directly extracted from soil’ (ISO TC4/WG4) - Intermediary meeting of ISO (Berlin, Mar

2012) - Annual meeting of ISO (Helsinki, Sep 2012)

Organization of the International interaboratory ring test - first call for ring test published by ISO

the 11 December 2012.

1. Martin-Laurent F, Kandeler E, Petric I, Djuric S, Karpouzas D (2013) ECOFUN-MICROBIODIV: an FP7 European project for developing and evaluating innovative tools for assessing the impact of pesticides on soil functional microbial diversity: towards new pesticide registration regulation? Env. Sci. Poll. Res. 2: 1203-1205

2. Karpouzas D, Papadopoulou E, Ipsilantis I, Petric I, Udikovic-Kolic N, Djuric S.,Kandeler E, Menkissoglu-Spiroudi U, Martin-Laurent F (2013) Potential side effects of nicosulfuron on abundance and diversity of arbuscular mycorrhizal fungi - towards the establishment of a pesticide soil microbial ecotoxicity indicator. Submitted to Ecological Indicators

3. Karpouzas D, Kandeler E, Bru D, Kramer S, Djuric S, Martin-Laurent F (2013) Laboratory and field assessment of the impact of nicosulfuron on the function and the abundance of soil microbes using a multi-methodological approach. In preparation for Soil Biology and Biochemistry

4. Petric I, Udikovic-Kolic N, Karpouzas D, Kandeler E, Djuric S, Bru, D, Martin-Laurent F (2013) Adaptation of the soil microbial community to the herbicide nicosulfuron exposure: a dose effect study. In preparation for Pest Management Science

Publications in International Journal


Thank you for your attention

Pr E Kandeler, Auer Y., Kramer S. Uni Hohenheim, Hohenheim

D Karpouzas, E Papadopoulou, I Ipsilantis, U Menkissoglu-Spiroudi; Uni of Thessaly, Thessaly

S Djuric, Faculty of Agriculture, Novi-Sad

F Martin-Laurent, D Bru, I Friedel INRA, Dijon

N Udiković-Kolić, Rudjer Boskovic Institute, Zagreb


https://www.uni-hohenheim.de/startseite.html?&L=1

http://www.uth.gr/en

http://www.uns.ac.rs/en/prozor.htm

Documents

ECOFUN-MICROBIODIV: an FP7 European project for developing ... 11-50.pdf · pesticides impact on the on soil microbial communities and do not reveal consequences on soil ecosystem