Micro PIXE mapping proves a differential distribution and concentration of trace elements in fungal structures of Rhizophagus intraradices.

Arbuscular mycorrhizal (AM) fungi can sequester different potentially toxic elements, such as trace elements (TEs), within their structures to alleviate the toxicity for its host plant and themselves. To elucidate the role of AM fungi in TEs immobilization in the rhizosphere of host plants, it is important to know the TEs distribution in AM fungal structures. In the present study, we investigated the distribution and concentration of TEs within extraradical spores and mycelium of the AM fungus Rhizophagus intraradices, collected from the rhizosphere of Senecio bonariensis plants grown in a soil polluted with multiple TEs, by using Particle-Induced X-ray Emission with a micro-focused beam (micro PIXE). This technique enabled the simultaneous micrometric mapping of elements in a sample. The calculated values were compared with those in the polluted substrate, measured by the Wavelength Dispersive X-ray Fluorescence technique. The highest concentrations of Fe, P, Ti, Mn, Cr, Cu and Zn were found in AM fungal spores, where they were accumulated, while extraradical mycelium was enriched in Cu. Finally, we demonstrated that AM fungi can simultaneously accumulate high amounts of different TEs in their structures, thus reducing the toxicity of these elements to its host plant.

Fungal extracellular phosphatases: their role in P cycling under different pH and P sources availability.

AIMS: The aim of this work is to analyse the effect of pH, fungal identity and P chemical nature on microbial development and phosphatase release, discussing solubilization and mineralization processes in P cycling. METHODS AND RESULTS: P solubilizing fungi (Talaromyces flavus, T. helicus L, T. helicus N, T. diversus and Penicillium purpurogenum) were grown under three pH conditions (6, 6·5 and 8·5) and with different inorganic (calcium, iron, aluminium and rock) and organic (lecithin and phytate) P sources. P solubilization, mineralization, growth and phosphatase production were recorded. Acid and neutral environments maximized fungal development and P recycling. P chemical nature changed the phosphatases release pattern depending on the fungal identity. Acid phosphatase activity was higher than alkaline phosphatases, regardless of pH or sample times. Alkaline phosphatases were affected by a combination of those factors. CONCLUSIONS: P chemical nature and pH modify fungal growth, P mineralization and solubilization processes. The underlying fungal identity-dependent metabolism governs the capacity and efficiency of P solubilization and mineralization. P solubilization and mineralization processes are interrelated and simultaneously present in soil fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: This study constitutes a reference work to improve the selection of fungal bioinoculants in different environmental conditions, highlighting their role in P cycling.

Carbon and Nitrogen Sources Influence Tricalcium Phosphate Solubilization and Extracellular Phosphatase Activity by Talaromyces flavus.

The aim of this work was to study phosphate (P) solubilization (and the processes involved in this event) by Talaromyces flavus (BAFC 3125) as a function of carbon and/or nitrogen sources. P solubilization was evaluated in NBRIP media supplemented with different carbon (glucose, sorbitol, sucrose, and fructose) and nitrogen (L-asparagine, urea, ammonium sulfate (AS), and ammonium nitrate (AN) combinations. The highest P solubilization was related to the highest organic acid production (especially gluconic acid) and pH drop for those treatments where glucose was present. Also P solubilization was higher when an inorganic nitrogen source was supplemented to the media when compared to an organic one. Although not being present an organic P source, phosphatase activity was observed. This shows that P mineralization and P solubilization can occur simultaneously, and that P mineralization is not induced by the enzyme substrate. The combination that showed highest P solubilization was for AN-glucose. The highest acid phosphatase activity was for AS-fructose, while for alkaline phosphatase were for AS-fructose and AN-fructose. Acid phosphatase activity was higher than alkaline. P solubilization and phosphatase activity (acid and alkaline) were influenced by the different carbon-nitrogen combinations. A better understanding of phosphate-solubilizing fungi could bring a better use of soil P.

Diversity of arbuscular mycorrhizal fungi in soil from the Pampa Ondulada, Argentina, assessed by pyrosequencing and morphological techniques.

The aim of this study was to assess the effects of agronomic practices on the arbuscular mycorrhizal (AM) fungal community in soils from the Pampa Ondulada region (Argentina), and to compare conclusions reached when using pyrosequencing or a morphological approach. The AM fungal diversity of 3 agricultural exploitations located in the Pampa Ondulada region (Argentina) was assessed by using 454 amplicon pyrosequencing and morphological (based on spore traits) approaches. Two kinds of soil managements are found in these sites: agronomic and non-agronomic. A total of 188 molecular operational taxonomic units and 29 morphological species of AM fungi were identified. No effect of soil management on AM richness was detected. AM fungal communities were more diverse and equitable in the absence of agronomic management. In contrast, the results on ß-diversity varied according to the methodology used. We concluded that agronomic management of soil has a negative effect on AM fungal community biodiversity in the Pampa Ondulada region. We also conclude that both methodologies complement each other in the study of AM fungal ecology. This study greatly improved the knowledge about AM fungi in South America where the molecular diversity of AM fungi was practically unknown.

Clonostachys rosea BAFC3874 as a Sclerotinia sclerotiorum antagonist: mechanisms involved and potential as a biocontrol agent.

AIMS: To establish the modes of action of the antagonistic fungal strain Clonostachys rosea BAFC3874 isolated from suppressive soils against Sclerotinia sclerotiorum and to determine its potential as a biocontrol agent. METHODS AND RESULTS: The antagonistic activity of C. rosea BAFC3874 was determined in vitro by dual cultures. The strain effectively antagonized S. sclerotiorum in pot-grown lettuce and soybean plants. Antifungal activity assays of C. rosea BAFC3874 grown in culture established that the strain produced antifungal compounds against S. sclerotiorum associated with secondary metabolism. High mycelial growth inhibition coincided with sclerotia production inhibition. The C. rosea strain produced a microheterogeneous mixture of peptides belonging to the peptaibiotic family. Moreover, mycoparasitism activity was observed in the dual culture. CONCLUSIONS: Clonostachys rosea strain BAFC3874 was proved to be an effective antagonist against the aggressive soil-borne pathogen S. sclerotiorum in greenhouse experiments. The main mechanisms involve peptaibiotic metabolite production and mycoparasitism activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Clonostachys rosea BAFC3874 may be a good fungal biological control agent against S. sclerotiorum. In addition, we were also able to isolate and identify peptaibols, an unusual family of compounds in this genus of fungi.

Medium pH, carbon and nitrogen concentrations modulate the phosphate solubilization efficiency of Penicillium purpurogenum through organic acid production.

AIMS: To study phosphate solubilization in Penicillium purpurogenum as function of medium pH, and carbon and nitrogen concentrations. METHODS AND RESULTS: Tricalcium phosphate (CP) solubilization efficiency of P. purpurogenum was evaluated at acid or alkaline pH using different C and N sources. Glucose- and (NH(4) )(2) SO(4) -based media showed the highest P solubilization values followed by fructose. P. purpurogenum solubilizing ability was higher in cultures grown at pH 6·5 than cultures at pH 8·5. Organic acids were detected in both alkaline and neutral media, but the relative percentages of each organic acid differed. Highest P release coincided with the highest organic acids production peak, especially gluconic acid. When P. purpurogenum grew in alkaline media, the nature and concentration of organic acids changed at different N and C concentrations. A factorial categorical experimental design showed that the highest P-solubilizing activity, coinciding with the highest organic acid production, corresponded to the highest C concentration and lowest N concentration. CONCLUSIONS: The results described in the present study show that medium pH and carbon and nitrogen concentrations modulate the P solubilization efficiency of P. purpurogenum through the production of organic acids and particularly that of gluconic acid. In the P solubilization optimization studies, glucose and (NH(4) )(2) SO(4) as C and N sources allowed a higher solubilization efficiency at high pH. SIGNIFICANCE AND IMPACT OF THE STUDY: This organism is a potentially proficient soil inoculant, especially in P-poor alkaline soils where other P solubilizers fail to release soluble P. Further work is necessary to elucidate whether these results can be extrapolated to natural soil ecosystems, where different pH values are present. Penicillium purpurogenum could be used to develop a bioprocess for the manufacture of phosphatic fertilizer with phosphate calcium minerals.

Cyclosporine A from a nonpathogenic Fusarium oxysporum suppressing Sclerotinia sclerotiorum.

AIMS: To evaluate the antagonistic activity of Fusarium oxysporum nonpathogenic fungal strain S6 against the phytopathogenic fungus Sclerotinia sclerotiorum and to identify the antifungal compounds involved. METHODS AND RESULTS: The antagonistic activity of Fusarium oxysporum strain S6 was determined in vitro by dual cultures. The metabolite responsible for the activity was isolated by chromatographic techniques, purified and identified by spectroscopic methods as cyclosporine A. The antifungal activity against the pathogen was correlated with the presence of this metabolite by a dilution assay and then quantified. Cyclosporine A caused both growth inhibition and suppression of sclerotia formation. In a greenhouse assay, a significant increase in the number of surviving soybean (Glycine max) plants was observed when S. sclerotiorum and F. oxysporum (S6) were inoculated together when compared with plants inoculated with S. sclerotiorum alone. CONCLUSION: Fusarium oxysporum (S6) may be a good fungal biological control agent for S. sclerotiorum and cyclosporine A is the responsible metabolite involved in its antagonistic activity in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Cyclosporine A has not been previously described as an inhibitor of S. sclerotiorum. Its minimum inhibitory concentration (MIC) of 0.1 microg disc(-1) makes it suitable to use as a biofungicide. In vivo experiments showed that F. oxysporum (S6) is a good candidate for the biocontrol of S. sclerotiorum in soybean.

Histopathology of Sclerotinia sclerotiorum attack on flower parts of Helianthus annuus heads in tolerant and susceptible varieties.

Sunflower head rot is a major disease caused by Sclerotinia sclerotiorum. Sunflower varieties which are tolerant to the fungus have been developed. The changes occurring in flower parts at different times after inoculation with pathogen ascospores were studied for two sunflower varieties (tolerant HA 302 and susceptible HA 89). In variety HA 302 there was cell collapse, changes in cell wall composition, and an increase in phenolic compounds in the tissues of corolla and style, which prevented the pathogen from advancing. This response was weaker in susceptible variety HA 89, and occurred only in the style, so did not stop the pathogen from developing and reaching the ovary. Phenolic compounds were found in HA 302 corolla and style tissues only when the pathogen was present, constituted an induced response that prevented further development of the fungus. Principal component analysis (PCA) showed that at the beginning of the infection there was no difference in behavior between the two varieties. The difference arose during the final observation times, when in variety HA 89, the pathogen colonized ovary, style and base of filaments and produced noticeable colonization of the corolla.

Fungal populations on sunflower (Helianthus annuus) anthosphere and their relation to susceptibility or tolerance to Sclerotinia sclerotiorum attack.

Analysis of the fungal flora from different floret parts of various sunflower (Helianthus annuus) varieties showed that there are differences in both fungal species and frequency, depending on whether the sunflower variety is susceptible (SV) or tolerant (TV) to attack of the flower heads by the ascomycete pathogen Sclerotinia sclerotiorum. The sunflower varieties analyzed were SV: HA 300 and Z 20028, and TV: HA 302, Z AV 8410 and Z 30629. The isolates showed different "in vitro" behavior as biocontrol agents. The most common types of interaction with Sclerotinia sclerotiorum were D2 and D2+ (hyphal contact) for isolates from SV and TV, while some of the isolates from TV displayed antibiosis. The microorganisms that colonize TV florets play a part in an indirect mechanism that protects flowers from ascospore germination and pathogen growth.

Effect of the fungicide benomyl on spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae.

The fungicide benomyl inhibited spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae when applied at doses of 21.25 microg/ml (agronomic dose), 10.62 microg/ml and 10 microg/ml. G. mosseae was able to germinate in the presence of 2.12 microg/ml of benomyl, and the percentage of spore germination was unaffected by dosis of 0.1, 0.01 and 0.001 microg/ml of the fungicide. However, all doses of fungicide tested in this study decreased the hyphal length. When ungerminated G. mosseae spores previously exposed to benomyl were transferred to water-agar medium without benomyl, the maximum germination was 16%. Small spores of G. mosseae were more resistant to benomyl than the larger ones. Our results show some of the factors which can explain the variability of the effect of benomyl on arbuscular mycorrhizal fungi.

[Evaluation of Trichoderma spp. as antagonist of Rhizoctonia solani in vitro and as biocontrol of greenhouse tomato plants]. / Evaluación de Trichoderma spp. como antagonista de Rhizoctonia solani in vitro y como biocontrolador del damping-off de plantas de tomate en invernadero.

Five Trichoderma isolates were compared in their ability for controlling Rhizoctonia solani attack to tomato plants in greenhouse and as antagonists of this pathogen in three independent laboratory assays. Four out of five isolates showed biocontrol ability and decreased pathogen growth and survival of its sclerotia in soil. Results suggest that dual cultures in Petri dishes and mycoparasitism assays against R. solani sclerotia may be useful for detecting isolates effective as biological control agents against this pathogen in tomato plants.

Evaluación de Trichoderma spp. como antagonista de Rhizoctonia solani in vitro y como biocontrolador del damping-off de plantas de tomate en invernadero / [Evaluation of Trichoderma spp. as antagonist of Rhizoctonia solani in vitro and as biocontrol of greenhouse tomato plants]

Five Trichoderma isolates were compared in their ability for controlling Rhizoctonia solani attack to tomato plants in greenhouse and as antagonists of this pathogen in three independent laboratory assays. Four out of five isolates showed biocontrol ability and decreased pathogen growth and survival of its sclerotia in soil. Results suggest that dual cultures in Petri dishes and mycoparasitism assays against R. solani sclerotia may be useful for detecting isolates effective as biological control agents against this pathogen in tomato plants.

Influence of the insecticide dimethoate on arbuscular mycorrhizal colonization and growth in soybean plants.

Application to the soil of the insecticide dimethoate had no effect on the growth of soybean colonized by the arbuscular mycorrhizal (AM) fungus Glomus mosseae and by the indigenous AM fungus. The application of the recommended concentration of dimethoate decreased the percentage of colonization of soybean by the indigenous AM population, but no significant effect was observed on the colonization of soybean inoculated with G. mosseae. The insecticide did not affect the germination of G. mosseae spores; however, 0.5 mg/l of dimethoate increased the germination of Gigaspora roseae and 5 mg/l of dimethoate decreased the germination of Scutellospora castaneae spores.

Evaluación de Trichoderma spp. como antagonista de Rhizoctonia solani in vitro y como biocontrolador del damping-off de plantas de tomate en invernadero. / [Evaluation of Trichoderma spp. as antagonist of Rhizoctonia solani in vitro and as biocontrol of greenhouse tomato plants]

Five Trichoderma isolates were compared in their ability for controlling Rhizoctonia solani attack to tomato plants in greenhouse and as antagonists of this pathogen in three independent laboratory assays. Four out of five isolates showed biocontrol ability and decreased pathogen growth and survival of its sclerotia in soil. Results suggest that dual cultures in Petri dishes and mycoparasitism assays against R. solani sclerotia may be useful for detecting isolates effective as biological control agents against this pathogen in tomato plants.

Biological control of Sclerotinia sclerotiorum attacking soybean plants. Degradation of the cell walls of this pathogen by Trichoderma harzianum (BAFC 742). Biological control of Sclerotinia sclerotiorum by Trichoderma harzianum.

Two experiments of biological control of Sclerotinia sclerotiorum, one in the greenhouse and the other in the field, were carried out with soybean and Trichoderma harzianum as host and antagonist, respectively. Significant control of disease was achieved in both experiments, but there were no significant differences in plant growths. In the greenhouse, the application of T. harzianum as alginate capsules, increased the survival of soybean plants more than 100% with respect to the disease treatment. In the field, T. harzianum treated plants survived 40% more than those from the disease treatment, showing a similar survival level to control plants. Besides, a significant reduction (62.5%) in the number of germinated sclerotia was observed in the Trichoderma treated plot. Chitinase and 1,3-beta- glucanase activities were detected when T. harzianum was grown in a medium containing Sclerotinia sclerotiorum cell walls as sole carbon source. In addition, electrophoretic profiles of proteins induced in T. harzianum showed quantitative differences between major bands obtained in the media induced by S. sclerotiorum cell walls and that containing glucose as a sole carbon source.

Interaction between Aspergillus niger van Tiegh. and Glomus mosseae. (Nicol. & Gerd.) Gerd. & Trappe.

Percent germination and length of hyphae of germinated Glomus mosseae spores, cultivated on water agar, decreased significantly in the presence of Aspergillus niger; this decrease was independent of any change in pH of the medium. Soluble and volatile compounds produced by A. niger significantly decreased percentage spore germination and the hyphal length of G. mosseae on water agar. The decrease caused by volatile compounds was significantly greater when A. niger was grown on malt extract agar. Shoot dry weights of maize and lettuce plants cultivated in soil in pots, and percentage arbuscular mycorrhizal (AM) root colonization of plants grown either in sand: vermiculite tubes inoculated with G. mosseae spores or in soil in pots with soil inoculum, were unaffected by A. niger when this saprobe was inoculated 2 wk after G. mosseae. Shoot dry weights and percentage AM colonization of plants decreased when the saprobic fungus was inoculated at the same time or 2 wk before G. mosseae. However, the metabolic activity resulting from AM colonization, measured as the percentage of mycelium showing succinate dehydrogenase activity, decreased in all treatments. The population of A. niger decreased when inoculated to the rhizosphere of plants at the same time as, or 2 wk after, G. mosseae, but not when it was inoculated 2 wk before G. mosseae. Our results show that G. mosseae decreases the saprobic fungal population through its effect on the plant, whereas A. niger, by the production of soluble or volatile substances, inhibits G. mosseae in its extramatrical stage.