Role of gibberellins during arbuscular mycorrhizal formation in tomato: new insights revealed by endogenous quantification and genetic analysis of their metabolism in mycorrhizal roots.

Gibberellins (GAs) are key regulators of plant growth and development and recent studies suggest also a role during arbuscular mycorrhizal (AM) formation. Here, complementary approaches have been used to obtain a clearer picture that correlates AM fungal development inside roots with GA metabolism. An extensive analysis of genes associated with GA metabolism as well as a quantification of GA content in roots was made. Application of GA3 and its biosynthesis inhibitor prohexadione calcium (PrCa) combined with a GA-constitutive response mutant (procera) were used to determine whether fungal colonization is altered by the level of these hormones or by changes in the GA-signaling pathway. The increased levels of specific GAs from the 13-hydroxylation pathway in mycorrhizal roots correlate closely with the increased expression of genes coding enzymes from the GA biosynthetic trail. The imbalance of GAs in tomato roots caused by exogenous applications of GA3 or PrCa affects arbuscules in both negative and positive ways, respectively. In addition, procera plants were adversely affected by the mycorrhization process. Our findings demonstrate that an imbalance in favor of an increased amount of GAs negatively affects the frequency of mycorrhization and particularly the arbuscular abundance in tomato mycorrhizal roots and the results point out that AM formation is associated with a change in the 13-hydroxylation pathway of GAs.

Evaluation of arbuscular mycorrhizal fungi capacity to alleviate abiotic stress of olive (Olea europaea L.) plants at different transplant conditions.

The capacity of roots to sense soil physicochemical parameters plays an essential role in maintaining plant nutritional and developmental functions under abiotic stress. These conditions generate reactive oxygen species (ROS) in plant tissues causing oxidation of proteins and lipids among others. Some plants have developed adaptive mechanisms to counteract such adverse conditions such as symbiotic association with arbuscular mycorrhizal fungi (AMF). AMF enhance plant growth and improve transplant survival by protecting host plants against environmental stresses. The aim of this study was to evaluate the alleviation of transplanting stress by two strains of Rhizophagus irregularis (GC2 and GA5) in olive. Our results show that olive plants have an additional energetic expense in growth due to an adaptative response to the growing stage and to the mycorrhizal colonization at the first transplant. However, at the second transplant the coinoculation improves olive plant growth and protects against oxidative stress followed by the GA5-inoculation. In conclusion, a combination of two AMF strains at the beginning of olive propagation produces vigorous plants successfully protected in field cultivation even with an additional cost at the beginning of growth.

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.

Solid state fermentation of olive mill residues by wood- and dung-dwelling Agaricomycetes: effects on peroxidase production, biomass development and phenol phytotoxicity.

The in vivo conversion of dry olive mill residue (DOR) by wood- and dung-dwelling fungi - Auricularia auricula-judae, Bjerkandera adusta and Coprinellus radians - increases peroxidase secretion up to 3.2-3.5-fold (∼1.3, 3.5 and 7.0 Ug(-1) DOR for dye-decolorizing peroxidase, manganese peroxidase and aromatic peroxygenases, respectively). The incubation of DOR with these fungi produced a sharp decrease in total phenolic content (100% within 4 wk), a reduction in phytotoxicity as well as a certain degree of plant growth caused by the stimulating effect of fungal-treated DOR. These findings correlate with a characteristic shift in the fragmentation pattern of water-soluble aromatics (detected at 280 nm) from low (0.2, 1.5 and 2.2 kDa, respectively) to high molecular mass (35 to >200 kDa), which demonstrates the presence of a polymerization process. Phenol-rich agricultural residues are a useful tool for enzyme expression and production studies of peroxidase-producing Agaricomycetes which could make DOR a valuable organic fertilizer.

Role of arbuscular mycorrhizal fungus Rhizophagus custos in the dissipation of PAHs under root-organ culture conditions.

Polycyclic aromatic hydrocarbons (PAHs) are one of the most common contaminants in soil. Arbuscular mycorrhizal (AM) fungi make host plants resistant to pollutants. This study aims to evaluate the impact of anthracene, phenanthrene and dibenzothiophene on the AM fungus Rhizophagus custos, isolated from soil contaminated by heavy metals and PAHs, under monoxenic conditions. We found a high level of tolerance in R. custos to the presence of PAHs, especially in the case of anthracene, in which no negative effect on AM-colonized root dry weight (root yield) was observed, and also a decrease in the formation of anthraquinone was detected. Increased PAH dissipation in the mycorrhizal root culture medium was observed; however, dissipation was affected by the level of concentration and the specific PAH, which lead us to a better understanding of the possible contribution of AM fungi, and in particular R. custos, to pollutant removal.

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, O(2)·(-) 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 O(2)·(-) generation and EC-POX's activities and protein oxidation, but enhanced SOD activity, lipid peroxidation and proline content. Fluorescence imaging showed that ADOR induced O(2)·(-) and H(2)O(2) accumulation in the roots. The increase in SOD and the decrease in EC-POX's activities might be involved in the enhancement of H(2)O(2) 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.

Late activation of the 9-oxylipin pathway during arbuscular mycorrhiza formation in tomato and its regulation by jasmonate signalling.

The establishment of an arbuscular mycorrhizal (AM) symbiotic interaction is a successful strategy for the promotion of substantial plant growth, development, and fitness. Numerous studies have supported the hypothesis that plant hormones play an important role in the establishment of functional AM symbiosis. Particular attention has been devoted to jasmonic acid (JA) and its derivates, which are believed to play a major role in AM symbiosis. Jasmonates belong to a diverse class of lipid metabolites known as oxylipins that include other biologically active molecules. Recent transcriptional analyses revealed up-regulation of the oxylipin pathway during AM symbiosis in mycorrhizal tomato roots and indicate a key regulatory role for oxylipins during AM symbiosis in tomato, particularly those derived from the action of 9-lipoxygenases (9-LOXs). Continuing with the tomato as a model, the spatial and temporal expression pattern of genes involved in the 9-LOX pathway during the different stages of AM formation in tomato was analysed. The effects of JA signalling pathway changes on AM fungal colonization were assessed and correlated with the modifications in the transcriptional profiles of 9-LOX genes. The up-regulation of the 9-LOX oxylipin pathway in mycorrhizal wild-type roots seems to depend on a particular degree of AM fungal colonization and is restricted to the colonized part of the roots, suggesting that these genes could play a role in controlling fungal spread in roots. In addition, the results suggest that this strategy of the plant to control AM fungi development within the roots is at least partly dependent on JA pathway activation.

Effect of a new thermal treatment in combination with saprobic fungal incubation on the phytotoxicity level of alperujo.

Byproducts generated from food industries, such as olive oil mills, have been studied to decrease harmful pollution and their environmental consequences. In this work, a new thermal pretreatment and saprobic fungal incubation to detoxify alperujo (two-phase olive mill waste) have been evaluated in view of its use as fertilizer in agriculture. The sequential use of both methods simplifies the thermal conditions and incubation times of the fungal treatment. Optimization of the thermal treatment from 150 to 170 °C for 45 and 15 min, respectively, reduced the incubation time with Coriolpsis rigida from 20 to 10 weeks needed to reduce phytotoxic effects on tomato plants. Therefore, the combination of thermal and biological treatments will allow the development of the potential benefits of alperujo to improve nutrients in agricultural soil.

Ethylene-dependent/ethylene-independent ABA regulation of tomato plants colonized by arbuscular mycorrhiza fungi.

We investigated the relationship between ABA and ethylene regulating the formation of the arbuscular mycorrhiza (AM) symbiosis in tomato (Solanum lycopersicum) plants and tried to define the specific roles played by each of these phytohormones in the mycorrhization process. We analysed the impact of ABA biosynthesis inhibition on mycorrhization by Glomus intraradices in transgenic tomato plants with an altered ethylene pathway. We also studied the effects on mycorrhization in sitiens plants treated with the aminoethoxyvinyl glycine hydrochloride (AVG) ethylene biosynthesis inhibitor and supplemented with ABA. In addition, the expression of plant and fungal genes involved in the mycorrhization process was studied. ABA biosynthesis inhibition qualitatively altered the parameters of mycorrhization in accordance with the plant's ethylene perception and ethylene biosynthesis abilities. Inhibition of ABA biosynthesis in wild-type plants negatively affected all the mycorrhization parameters studied, while tomato mutants impaired in ethylene synthesis only showed a reduced arbuscular abundance in mycorrhizal roots. Inhibition of ethylene synthesis in ABA-deficient sitiens plants increased the intensity of mycorrhiza development, while ABA application rescued arbuscule abundance in the root's mycorrhizal zones. The results of our study show an antagonistic interaction between ABA and ethylene, and different roles of each of the two hormones during AM formation. This suggests that a dual ethylene-dependent/ethylene-independent mechanism is involved in ABA regulation of AM formation.

The Rhizobium sp. strain NGR234 systemically suppresses arbuscular mycorrhizal root colonization in a split-root system of barley (Hordeum vulgare).

Nitrogen-fixing bacteria (rhizobia) form a nodule symbiosis with legumes, but also induce certain effects on non-host plants. Here, we used a split-root system of barley to examine whether inoculation with Rhizobium sp. strain NGR234 on one side of a split-root system systemically affects arbuscular mycorrhizal (AM) root colonization on the other side. Mutant strains of NGR234 deficient in Nod factor production (strain NGRΔnodABC), perception of flavonoids (strain NGRΔnodD1) and secretion of type 3 effector proteins (strain NGRΩrhcN) were included in this study. Inoculation resulted in a systemic reduction of AM root colonization with all tested strains. However, the suppressive effect of strain NGRΩrhcN was less pronounced. Moreover, levels of salicylic acid, an endogenous molecule related to plant defense, were increased in roots challenged with rhizobia. These data indicate that barley roots perceived NGR234 and that a systemic regulatory mechanism of AM root colonization was activated. The suppressive effect appears to be Nod factor independent, but enhanced by type 3 effector proteins of NGR234.

Biochemical and molecular characterization of Coriolopsis rigida laccases involved in transformation of the solid waste from olive oil production.

Two laccase isoenzymes were purified and characterized from the basidiomycete Coriolopsis rigida during transformation of the water-soluble fraction of "alpeorujo" (WSFA), a solid residue derived from the olive oil production containing high levels of toxic compounds. Zymogram assays of laccases secreted by the fungus growing on WSFA and WSFA supplemented with glucose showed two bands with isoelectric points of 3.3 and 3.4. The kinetic studies of the two purified isoenzymes showed similar affinity on 2,6-dimethoxyphenol and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), used as phenolic and non-phenolic model substrate, respectively. The molecular mass of both proteins was 66 kDa with 9% N-linked carbohydrate. Physico-chemical properties of the purified laccases from media containing WSFA were similar to those obtained from medium with glucose as the main carbon source. In-vitro studies performed with the purified laccases revealed a 42% phenol reduction of WSFA, as well as changes in the molecular mass distribution. These findings indicate that these laccases are involved in the process of transformation, via polymerization by the oxidation of phenolic compounds present in WSFA. A single laccase gene, containing an open reading frame of 1,488 bp, was obtained in PCR amplifications performed with cDNA extracted from mycelia grown on WSFA. The product of the gene shares 90% identity (95% similarity) with a laccase from Trametes trogii and 89% identity (95% similarity) with a laccase from Coriolopsis gallica. This is the first report on purification and molecular characterization of laccases directly involved in the transformation of olive oil residues.

Mycorrhization of the notabilis and sitiens tomato mutants in relation to abscisic acid and ethylene contents.

We examined whether the reduced mycorrhization of abscisic acid (ABA)-deficient tomato mutants correlates with their incapacity in ABA biosynthesis and whether this effect is dependent on ethylene production. The mycorrhization of notabilis and sitiens mutants, which have different ABA deficiencies and an excess of ethylene production, was analyzed. Comparative analysis of the ABA-deficient tomato mutants showed both quantitative and qualitative differences in the pattern of arbuscular mycorrhiza (AM) colonization between the two tomato mutant phenotypes. The sitiens mutant showed a great limitation in fungal colonization (mycorrhizal intensity and arbuscule formation) well correlated with their incapacity in ABA biosynthesis. The notabilis plants, which maintained normal ABA levels in roots under our experimental conditions, appeared to be less affected in their capacity for AM formation, and only a decrease in mycorrhizal intensity was noted at the end of the mycorrhization process. Blockage of ABA formation after tungstate application resulted in a reduction in mycorrhization of wild-type tomato plants. The transcript accumulation of the mycorrhiza-responsive LePT4 gene (tomato phosphate transporter) was clearly associated with the ABA content and mycorrhiza development in roots, as the tungstate treatment in wild-type plants and the inherent ABA deficiency in sitiens mutants led to a complete abolishment of their expression. Our results suggest that the decrease in arbuscular abundance in mycorrhizal sitiens roots is directly associated with their ABA biosynthesis deficiency, and the accumulation of ethylene, as a consequence of ABA deficiency in the mutants, primarily affects mycorrhizal intensity.

Xyloglucanases in the interaction between saprobe fungi and the arbuscular mycorrhizal fungus Glomus mosseae.

We studied the production of xyloglucanase enzymes of pea and lettuce roots in the presence of saprobe and arbuscular mycorrhizal (AM) fungi. The AM fungus Glomus mosseae and the saprobe fungi Fusarium graminearum, Fusarium oxysporum-126, Trichoderma harzianum, Penicillium chrysogenum, Pleurotus ostreatus and Aspergillus niger were used. G. mosseae increased the shoot and root dry weight of pea but not of lettuce. Most of the saprobe fungi increased the level of mycorrhization of pea and lettuce, but only P. chrysogenum and T. harzianum inoculated together with G. mosseae increased the dry weight of pea and lettuce respectively. The AM and saprobe fungi increased the production of xyloglucanases by plant roots. The level of xyloglucanase activities and the number of xyloglucanolytic isozymes in plants inoculated with G. mosseae and most of the saprobe fungi tested were higher than when both microorganisms were inoculated separately. The possible relationship between xylogucanase activities and the ability of AM and saprobe fungi to improve the dry weight and AM root colonization of plants was discussed.