Final Report Summary - REMEDIAM (Impact of polyaromatic hydrocarbons on arbuscular mycorrhizal fungi and biochemical and molecular mechanisms involved in plant protection and pollutant dissipation)
Hydrocarbures and polycyclic aromatic hydrocarbons (PAHs) are widely distributed in soil all over the world, and many of them are highly toxic, mutagenic and/or carcinogenic to humans (IARC, 2010). They also exhibit a detrimental effect on the flora and fauna of contaminated habitats (Eom et al., 2007; Maliszewska-Kordybach et al., 2007). Among the many PAHs congeners, the US-EPA listed 16 priority PAHs to eliminate from polluted soils, including benzo[a]pyrene (B[a]P), a high molecular weight PAHs. Owing to their great persistence in soils, their dissipation remains challenging.
Among the emergent remediation methods of hydrocarbures-polluted soils, the phytoremediation assisted by arbuscular mycorrhizal fungi (AMF) represents a cost-effective and environmental-friendly innovative approach. The AMF is an important ecological and economical group of soil fungi that establish symbiotic associations with the roots of the vast majority of terrestrial plants. Their origin dates back 460 million years ago suggesting their role in the colonization of land by plants (Redecker et al., 2000; Smith and Read, 2008). In this association, AMF obligatory acquire their carbon from the plant, in exchange of water and minerals (i.e. phosphorus and nitrogen). These soil fungi also enhance plant tolerance to biotic and abiotic stresses including PAHs. Indeed, Debiane et al. (2008, 2009) demonstrated that mycorrhizal colonization provided higher PAHs tolerance to the roots by reducing the oxidative stress and, in particular, by limiting the damages to membranes and genome.
Studies conducted on phytoremediation of PAHs by mycorrhized plants demonstrated that extraradical mycelium plays a key role in the immobilization/sequestration of various pollutants and as such may restrict the diffusion of the pollutant to the roots. Anthracene (a low molecular weight PAH) immobilization was observed in hyphae of R. irregularis, and this mechanism participated actively in the enhanced anthracene dissipation (Verdin et al., 2006). On the other hand, contradictory results were reported concerning the PAHs dissipation promoted by mycorrhizal symbiosis and standardized studies should be performed.
With the increase in number of PAHs-polluted sites, it is necessary to understand how these pollutants affect the morphology and physiology of AMF and how these root symbionts may represent an option for remediation. The present project aims to determine the impact of PAHs on AMF having different life history strategies, i.e. adapted to grow either under stable (K-strategists) or disturbed (r-strategists) environments. We postulate that PAHs may differently affect AMF r/K-strategists which in turn may (1) protect the plant from PAHs toxicity in a different way and (2) differently participate in the dissipation of these molecules.
To investigate this hypothesis, three successive questions corresponding to the three research objectives, were addressed:
1. do PAHs differently impact the morpho-anatomy and physiology of AMF r/K strategists?
2. do AMF r/K strategists differently affect the resistance of plants to PAHs and crude oil?
3. do r/K strategists differently affect oil dissipation?
To answer the hypothesis and achieve these objectives, a combination of original and well-designed in vitro and in vivo controlled experiments was conducted. We used monoxenic cultivation systems. These systems have noticeably improved our understanding of the symbiosis since they allow non-destructive observations of AMF (Fortin et al., 2002). In vivo controlled cultures of AMF are more in line with natural conditions of plants culture, and allow studies on plant non cultivable in in vitro conditions.
The results obtained within this project are as follows:
✓ B[a]P (a high molecular weight PAH) and more importantly diesel at low concentrations have a marked impact on the AMF fungal life cycle (i.e. spore germination, spore production). Dynamics of germination varied between Rhizophagus (R.) irregularis MUCL 43204 and MUCL 41833, R. clarus 46238 and Gigaspora sp. MUCL 52331, which could be explained by the edapho-climatic origin of the spores and the susceptibility of them to the pollutant.
✓ Depending on the hydrocarbure concentration applied, the pollutant has a more pronounced fungitoxic effect (high concentrations) than a fungistatic effect (low concentrations).
✓ The healing mechanisms (i.e. the mechanism by which a fungus is able to repair damage) of germinated spores and extraradical mycelium is impacted by B[a]P and diesel.
✓ Plant development is impacted by B[a]P and diesel even at very low concentrations.
✓ Whereas mycorrhized B[a]P-stressed M. truncatula produced a similar number of leaves than non-stressed plants, non-mycorrhized ones produced less leaves. Moreover, the dry weight of the plant was greater in mycorhized roots than in non-mycorrhized ones, demonstrating a protection effect of the presence of the AMF in case of stress.
✓ B[a]P and diesel do not impact direct P uptake by the extraradical mycelium of different AMF species R. irregularis MUCL 41833 and R. clarus MUCL 46238.
✓ B[a]P and diesel negatively impact P uptake by maize (Zea mays L.) and Medicago truncatula plants.
✓ The presence of the AMF R. irregularis MUCL 41833 protects maize and M. truncatula by stimulating P accumulation and transport in whole plant, especially in shoot part.
✓ In vitro cultivation of whole plant in bi-compartmented Petri dishes as well as semi semi-hydroponic cultivation system are suitable for short-term nutrient (P) dynamics of uptake by the plants/AMF.
All these results demonstrated an impact of hydrocarbures on plant as well as on different AMF strategists. However, even if their development was impacted, the AMF were still able to take up P under the concentrations tested. Importantly, AMF-colonized plants had a better growth and accumulated and transported P in higher extent that non-mycorrhized ones. This demonstrated a protection conferred by the presence of the AMF, via, among other, a better nutrition in P.
As a general conclusion, the results from REMEDIAM demonstrate that AMF-colonized plants are more resistant to hydrocarbon pollutants. As a consequence, AMF may represent an alternative approach to be used in phytoremediation strategies or in strategies oriented towards the re-vegetation with suitable plant-AMF associations. Interestingly, the current European investment (both private and public) in sustainable soil remediation strategies represent less than 10 million Euro (i.e. about 20 times less than that of the USA) but tend to increase. The potential European market for soil phytoremediation of toxic and radionuclide pollutants was estimated to be about 300 billion Euro. The results obtained in REMEDIAM may thus stimulate SME’s to include and consider these soil fungi in their strategy which overall will be profitable for the economy of Europe.
Among the emergent remediation methods of hydrocarbures-polluted soils, the phytoremediation assisted by arbuscular mycorrhizal fungi (AMF) represents a cost-effective and environmental-friendly innovative approach. The AMF is an important ecological and economical group of soil fungi that establish symbiotic associations with the roots of the vast majority of terrestrial plants. Their origin dates back 460 million years ago suggesting their role in the colonization of land by plants (Redecker et al., 2000; Smith and Read, 2008). In this association, AMF obligatory acquire their carbon from the plant, in exchange of water and minerals (i.e. phosphorus and nitrogen). These soil fungi also enhance plant tolerance to biotic and abiotic stresses including PAHs. Indeed, Debiane et al. (2008, 2009) demonstrated that mycorrhizal colonization provided higher PAHs tolerance to the roots by reducing the oxidative stress and, in particular, by limiting the damages to membranes and genome.
Studies conducted on phytoremediation of PAHs by mycorrhized plants demonstrated that extraradical mycelium plays a key role in the immobilization/sequestration of various pollutants and as such may restrict the diffusion of the pollutant to the roots. Anthracene (a low molecular weight PAH) immobilization was observed in hyphae of R. irregularis, and this mechanism participated actively in the enhanced anthracene dissipation (Verdin et al., 2006). On the other hand, contradictory results were reported concerning the PAHs dissipation promoted by mycorrhizal symbiosis and standardized studies should be performed.
With the increase in number of PAHs-polluted sites, it is necessary to understand how these pollutants affect the morphology and physiology of AMF and how these root symbionts may represent an option for remediation. The present project aims to determine the impact of PAHs on AMF having different life history strategies, i.e. adapted to grow either under stable (K-strategists) or disturbed (r-strategists) environments. We postulate that PAHs may differently affect AMF r/K-strategists which in turn may (1) protect the plant from PAHs toxicity in a different way and (2) differently participate in the dissipation of these molecules.
To investigate this hypothesis, three successive questions corresponding to the three research objectives, were addressed:
1. do PAHs differently impact the morpho-anatomy and physiology of AMF r/K strategists?
2. do AMF r/K strategists differently affect the resistance of plants to PAHs and crude oil?
3. do r/K strategists differently affect oil dissipation?
To answer the hypothesis and achieve these objectives, a combination of original and well-designed in vitro and in vivo controlled experiments was conducted. We used monoxenic cultivation systems. These systems have noticeably improved our understanding of the symbiosis since they allow non-destructive observations of AMF (Fortin et al., 2002). In vivo controlled cultures of AMF are more in line with natural conditions of plants culture, and allow studies on plant non cultivable in in vitro conditions.
The results obtained within this project are as follows:
✓ B[a]P (a high molecular weight PAH) and more importantly diesel at low concentrations have a marked impact on the AMF fungal life cycle (i.e. spore germination, spore production). Dynamics of germination varied between Rhizophagus (R.) irregularis MUCL 43204 and MUCL 41833, R. clarus 46238 and Gigaspora sp. MUCL 52331, which could be explained by the edapho-climatic origin of the spores and the susceptibility of them to the pollutant.
✓ Depending on the hydrocarbure concentration applied, the pollutant has a more pronounced fungitoxic effect (high concentrations) than a fungistatic effect (low concentrations).
✓ The healing mechanisms (i.e. the mechanism by which a fungus is able to repair damage) of germinated spores and extraradical mycelium is impacted by B[a]P and diesel.
✓ Plant development is impacted by B[a]P and diesel even at very low concentrations.
✓ Whereas mycorrhized B[a]P-stressed M. truncatula produced a similar number of leaves than non-stressed plants, non-mycorrhized ones produced less leaves. Moreover, the dry weight of the plant was greater in mycorhized roots than in non-mycorrhized ones, demonstrating a protection effect of the presence of the AMF in case of stress.
✓ B[a]P and diesel do not impact direct P uptake by the extraradical mycelium of different AMF species R. irregularis MUCL 41833 and R. clarus MUCL 46238.
✓ B[a]P and diesel negatively impact P uptake by maize (Zea mays L.) and Medicago truncatula plants.
✓ The presence of the AMF R. irregularis MUCL 41833 protects maize and M. truncatula by stimulating P accumulation and transport in whole plant, especially in shoot part.
✓ In vitro cultivation of whole plant in bi-compartmented Petri dishes as well as semi semi-hydroponic cultivation system are suitable for short-term nutrient (P) dynamics of uptake by the plants/AMF.
All these results demonstrated an impact of hydrocarbures on plant as well as on different AMF strategists. However, even if their development was impacted, the AMF were still able to take up P under the concentrations tested. Importantly, AMF-colonized plants had a better growth and accumulated and transported P in higher extent that non-mycorrhized ones. This demonstrated a protection conferred by the presence of the AMF, via, among other, a better nutrition in P.
As a general conclusion, the results from REMEDIAM demonstrate that AMF-colonized plants are more resistant to hydrocarbon pollutants. As a consequence, AMF may represent an alternative approach to be used in phytoremediation strategies or in strategies oriented towards the re-vegetation with suitable plant-AMF associations. Interestingly, the current European investment (both private and public) in sustainable soil remediation strategies represent less than 10 million Euro (i.e. about 20 times less than that of the USA) but tend to increase. The potential European market for soil phytoremediation of toxic and radionuclide pollutants was estimated to be about 300 billion Euro. The results obtained in REMEDIAM may thus stimulate SME’s to include and consider these soil fungi in their strategy which overall will be profitable for the economy of Europe.