Mercedes García-Sánchez

Defence response of tomato seedlings to oxidative stress induced by phenolic compounds from dry olive mill residue

ADOR is an aqueous extract obtained from the dry olive mill residue (DOR) which contains the majority of its soluble phenolic compounds, which are responsible for its phytotoxic properties. Some studies have shown that ADOR negatively affects seed germination. However, to date, few studies have been carried out on the effect of ADOR on the oxidative stress of the plant. It is well known that saprobe fungi can detoxify these phenolic compounds and reduce the potential negative effects of ADOR on plants. To gain a better understanding of the phytotoxic effects and oxidative stress caused by this residue, tomato seeds were germinated in the presence of ADOR, treated and untreated with Coriolopsis rigida, Trametes versicolor, Pycnoporus cinnabarinus and Penicillium chrysogenum-10 saprobe fungi. ADOR sharply reduced tomato seed germination and also generated high levels of malondialdehyde (MDA), O(2)(-) and H(2)O(2). However, bioremediated ADOR did not negatively affect germination and reduced MDA, O(2)(-) and H(2)O(2) content in different ways depending on the fungus used. In addition, the induced defense response was studied by analyzing the activity of both antioxidant enzymes (superoxide dismutase (SOD), catalase, ascorbate peroxidasa, glutathione reductase (GR), peroxidases and coniferil alcohol peroxidasa) and detoxification enzymes (glutathione-S-transferase (GST)). Our findings suggest that, because ADOR is capable of inducing oxidative stress, tomato seedlings trigger a defense response through SOD, GR, and GST activity and through antioxidant and lignification processes. On the other hand, the bioremediation of ADOR plays an important role in counteracting the oxidative stress induced by the untreated residue.

Changes in soil microbial community functionality and structure in a metal-polluted site: The effect of digestate and fly ash applications

Soil from Trhové Dušníky (Příbram, Czech Republic) is characterized by its high polymetallic accumulations in Pb-Ag-Zn due to mining and smelting activities. In previous studies performed in our research group, we have evaluated the potential use of amendments that would reduce the mobility and availability of metals such as Hg. We have observed that the application of digestate and fly ash in metal-polluted soil has an impact in immobilizing these metals. However, until now we have lacked information about the effect of these amendments on soil microbial functionality and communities. The multi-contaminated soil was used to grow wheat in a pot experiment to evaluate the impact of digestate and fly ash application in soil microbial communities. Soil samples were collected after 30 and 60 days of treatment. The digestate application improved chemical attributes such as the content in total organic carbon (TOC), water soluble carbon (WSOC), total soluble carbon (C), total soluble nitrogen (N), and inorganic N forms (NO3(-)) as consequence of high content in C and N which is contained in digestate. Likewise, microbial activity was greatly enhanced by digestate application, as was physiological diversity. Bacterial and fungal communities were increased, and the microbial biomass was highly enhanced. These effects were evident after 30 and 60 days of treatment. In contrast, fly ash did not have a remarkable effect when compared to digestate, but soil microbial biomass was positively affected as a consequence of macro- and micro-nutrient sources applied by the addition of fly ash. This study indicates that digestate can be used successfully in the remediation of metal-contaminated soil.

Arbuscular mycorrhizal fungi alleviate oxidative stress induced by ADOR and enhance antioxidant responses of tomato plants.

The behaviour of tomato plants inoculated with arbuscular mycorrhizal (AM) fungi grown in the presence of aqueous extracts from dry olive residue (ADOR) was studied in order to understand how this symbiotic relationship helps plants to cope with oxidative stress caused by ADOR. The influence of AM symbiosis on plant growth and other physiological parameters was also studied. Tomato plants were inoculated with the AM fungus Funneliformis mosseae and were grown in the presence of ADOR bioremediated and non-bioremediated by Coriolopsis floccosa and Penicillium chrysogenum-10. The antioxidant response as well as parameters of oxidative damage were examined in roots and leaves. The data showed a significant increase in the biomass of AM plant growth in the presence of ADOR, regardless of whether it was bioremediated. The establishment and development of the symbiosis were negatively affected after plants were exposed to ADOR. No differences were observed in the relative water content (RWC) or PS II efficiency between non-AM and AM plants. The increase in the enzymatic activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6) and glutathione-S-transferase (GST; EC 2.5.1.18) were simultaneous to the reduction of MDA levels and H2O2 content in AM root growth in the presence of ADOR. Similar H2O2 levels were observed among non-AM and AM plants, although only AM plants showed reduced lipid peroxidation content, probably due to the involvement of antioxidant enzymes. The results highlight how the application of both bioremediated ADOR and AM fungi can alleviate the oxidative stress conditions, improving the growth and development of tomato plants.

The effects of the arbuscular mycorrhizal fungus Glomus deserticola on growth of tomato plants grown in the presence of olive mill residues modified by treatment with saprophytic fungi.

Olive oil extraction generates large amounts of olive mill residues (DOR) which may be used as fertilizer. The influence of arbuscular mycorrhizal (AM) on the phytotoxicity of dry olive residue (DOR) transformed with saprophytic fungi was studied. Aqueous extraction of DOR gave an (ADOR) fraction and an exhausted (SDOR) fraction, both of which had less phytotoxicity for tomato than the original DOR. The saprophytic fungiTrametes versicolor andPycnoporus cinnabarinus further decreased the phytotoxicity of ADOR and SDOR on tomato. The decrease of phenols concentration and the differences in the level of laccase activity caused by these fungi suggest did not account fully for the reduced phytoxicity but the fact that the higher hydrolytic enzyme activity ofP. cinnabarinus, paralleled the decrease of phytotoxicity, indicates that these enzymes seem to be involved. The AM fungusGlomus deserticola increased or exacerbated the beneficial effect of SDOR incubated with saprophytic fungi, in terms of dry weight of tomato plants. The percentage of root length colonized byG. deserticola strongly decreased in presence of DOR, but the level of mycorrhization was higher in presence of ADOR or SDOR. Our results suggest that the combination of aqueous extraction and incubation with saprophytic fungi will open the way for the use of olive oil extraction residues as organic amendment in agricultural soils.

A comparative study to evaluate natural attenuation, mycoaugmentation, phytoremediation, and microbial-assisted phytoremediation strategies for the bioremediation of an aged PAH-polluted soil

Biological treatments are considered an environmentally option to clean-up polluted soil with polycyclic aromatic hydrocarbons (PAHs). A pot experiment was conducted to comparatively evaluate four different strategies, including natural attenuation (NA), mycoaugmentation (M) by using Crucibulum leave, phytoremediation (P) using maize plants, and microbial-assisted phytoremediation (MAP) for the bioremediation of an aged PAH-polluted soil at 180 days. The P treatment had higher affinity degrading 2-3 and 4 ring compounds than NA and M treatments, respectively. However, M and P treatments were more efficient in regards to naphthalene, indeno[l,2,3-c,d]pyrene and benzo[g,h,i]perylene degradation respect to NA. However, 4, 5-6 rings undergo a strong decline during the microbe-assisted phytoremediation, being the treatment which determined the highest rates of PAHs degradation. Sixteen PAH compounds, except fluorene and dibenzo[a,h]anthracene, were found in maize roots, whereas the naphthalene, phenanthrene, anthracene, fluoranthene, and pyrene were accumulated in the shoots, in both P and MAP treatments. However, higher PAH content in maize biomass was achieved during the MAP treatment respect to P treatment. The bioconversion and translocation factors were less than 1, indicating that phystabilization/phytodegradation processes occurred rather than phytoextraction. The microbial biomass, activity and ergosterol content were significantly boosted in the MAP treatment respect to the other treatments at 180 days. Ours results demonstrated that maize-C. laeve association was the most profitable technique for the treatment of an aged PAH-polluted soil when compared to other bioremediation approaches.

Stabilization of different starting materials through vermicomposting in a continuous-feeding system: Changes in chemical and biological parameters

In this study the feasibility of Eisenia andrei to digest great amount of wastes including horse manure (HM), apple pomace (AP), grape pomace (GP), and digestate (DG) was monitored through a continuous-feeding system. New layers of fresh material were gradually added to form an aged-profile of layers in order to understand the interaction between earthworms and microorganisms during vermicomposting. Thus, changes in chemical and biological parameters were evaluated for 240days. The earthworm population reached maximum values in 120 d-old-layer, which was related to an increase in overall microbial biomass, assayed as dehydrogenase activity, in all of the processed materials. The pH was generally alkaline or neutral in all of the materials. The electrical conductivity did not modify significantly during vermicomposting, except in the case of the processed GP, and DG. The stabilization, in all of the processed materials, was detected after 240 d of vermicomposting, as indicated the decline in the content of dissolved organic carbon (DOC). The N-NO3- content exhibited an enhanced in the processed HM and AP, while a generalized decreased was found in the GP, and DG materials in 240 d-old-layer. The decline in microbial biomass activity, in all processed substrates, was related to a decrease in the earthworm activity after 240 d of vermicomposting, indicating a high degree of stabilization. However, the β-glucosidase, phosphatase, protease, and o-diphenol oxidase activities were different according to the age of layers and type of processed material. The phytotoxicity test indicated that the end products of the processed AP and DG were chemically stable and enriched with nutrients in comparison with the HM and GP vermicompost. This fact indicates to stabilization (maturation) in the end product, which is important for its safe disposal as an organic nutrient-rich product.

Applications of organic and inorganic amendments induce changes in the mobility of mercury and macro- and micronutrients of soils.

Both soil organic matter and sulfur (S) can reduce or even suppress mercury (Hg) mobility and bioavailability in soil. A batch incubation experiment was conducted with a Chernozem and a Luvisol artificially contaminated by 440 mg·kg−1 Hg showing wide differences in their physicochemical properties and available nutrients. The individual treatments were (i) digestate from the anaerobic fermentation of biowaste; (ii) fly ash from wood chip combustion; and (iii) ammonium sulfate, and every treatment was added with the same amount of S. The mobile Hg portion in Chernozem was highly reduced by adding digestate, even after 1 day of incubation, compared to control. Meanwhile, the outcome of these treatments was a decrease of mobile Hg forms as a function of incubation time whereas the contents of magnesium (Mg), potassium (K), iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), and phosphorus (P) were stimulated by the addition of digestate in both soils. The available calcium (Ca) contents were not affected by the digestate addition. The experiment proved digestate application as the efficient measure for fast reduction of mobile Hg at extremely contaminated soils. Moreover, the decrease of the mobile mercury portion was followed by improvement of the nutrient status of the soils.

Oxidative stress induced in sunflower seedling roots by aqueous dry olive-mill residues.

The contamination of soils with dry olive-mill residue can represent a serious problem as being an environmental stressor in plants. It has been demonstrated that inoculation of aqueous extract of olive oil-mill residue (ADOR) with saprobe fungi removes some phenolic compounds. In this paper we studied the effect of ADOR uninoculated or inoculated with saprobe fungi in sunflower seedling roots. The germination and root growth, O2·- generation, superoxide dismutase (SOD) and extracellular peroxidases (EC-POXs) activities, and the content of some metabolites involved in the tolerance of stress were tested. The roots germinated in ADOR uninoculated show a decrease in meristem size, resulting in a reduction of the root length and fresh weight, and in the number of layers forming the cortex, but did not alter the dry weight, protein and soluble amino acid content. ADOR caused the decreases in O2·- generation and EC-POX′s activities and protein oxidation, but enhanced SOD activity, lipid peroxidation and proline content. Fluorescence imaging showed that ADOR induced O2·- and H2O2 accumulation in the roots. The increase in SOD and the decrease in EC-POX′s activities might be involved in the enhancement of H2O2 content and lipid peroxidation. Control roots treated with ADOR for 10 min show an oxidative burst. Roots germinated in ADOR inoculated with saprobe fungi partially recovered normal levels of ROS, morphological characteristics and antioxidant activities. These results suggested that treatment with ADOR caused a phytotoxic effect during germination inducing an oxidative stress. The inoculation of ADOR with saprobe fungi limited the stress.

Suppressive effect of olive residue and saprophytic fungi on the growth of Verticillium dahliae and its effect on the dry weight of tomato (Solanum lycopersicum L.)

The saprophytic fungi Aspergillus niger, Coriolopsis rigida, Fusarium lateritium, F. oxysporum, Mucor racemosus, Paecilomyces farinosus, Penicillium chrysogenum, P. restrictum, Trametes versicolor, Trichoderma harzianum, T. pseudokoningii and T. viride were able to decrease the growth in vitro of Verticillium dahlie in the presence of aqueous extract of olive residue. The conidia number of V. dahliae decreased when grown on aqueous extract of olive residue, autoclaved or filtered through 0.45 micron filters after culture of the fungi. These results suggest not only the predominance of suppressive substances of a biological nature, but also the existence of non-biological inhibitory substances. The olive residue decreased the negative effect of V. dahliae on shoot and root dry weight of tomato (Solanum lycopersicum L.), by the antifungal compounds present in the olive residue and by the antifungal substances produced by the antagonistic saprophytic fungi grown in this residue.

Reduced dry olive residue phytotoxicity in the field by the combination of physical and biological treatments

Olive oil extraction generates large amounts of olive mill residues (DOR) which may be used as organic fertilizer. The influence of a combination of physical fractionation and saprobe fungal incubation on the phytotoxicity of DOR was studied. The physical fractions of DOR, obtained following extraction using ethyl acetate (EDOR) and wa- ter (ADOR) were less phytotoxic than DOR with respect to the shoot dry weight of to- mato. There was no relationship between the total phenol content of the different DOR physical fractions and their phytotoxicity. The saprophytic fungus Coriolopsis rigida reduced DOR and ADOR phytotoxicity and eliminated the phytotoxicity of EDOR. However, unlike the physical treatments of DOR, the decrease in the phenol content of EDOR and ADOR caused by C. rigida was closely paralleled to the decrease in their phytotoxicity. After 30 days of incubation, C. rigida was able to eliminate the phytotoxicity of EDOR on tomato plants grown in the field.