Survey of organochlorine-tolerant culturable mycota from contaminated soils, and 2,4-D removal ability of Penicillium species in synthetic wastewater.

Agrochemical wastewater, which is produced by the extensive use of herbicides, has become a serious environmental pollutant. In this study, culturable mycota were isolated from soils contaminated with herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA), and their ability to tolerate and remove 2,4-D was assessed. The mycota were isolated on solid medium supplemented with 10 mmol L-1 of MCPA or 2,4-D. Tolerance and removal assays were performed in synthetic wastewater, and removal was quantified by HPLC-UV and MS/MS. Fusarium spp., Aspergillus spp., and Penicillium spp. were the most frequently isolated genera. Six Penicillium strains were able to tolerate up to 25 mmol L-1 of 2,4-D. Within this group, two P. crustosum strains (RCP4 and RCP13) degraded more than 50% of the 2,4-D in the medium during the first 7 days of incubation. Removal percentages reached 54% for RCP4 and 75% for RCP13 after 14 days. These two strains, therefore, could potentially be considered for the design of bioaugmentation strategies aimed at reducing contamination by 2,4-D in wastewater.

Fungal biodegradation of chlorinated herbicides: an overview with an emphasis on 2,4-D in Argentina.

Chlorinated herbicides are one of the main types of pesticide used in agriculture. In Argentina, 2,4-dichlorophenoxyacetic acid (2,4-D) is the most applied herbicide for the control of broadleaf weeds, but the risks it poses for the environment and human health are cause for great concern. A promising technology to remove this kind of pollutants, or neutralize them in such a way that they become less or non-toxic, is the use of degrading or detoxifying microorganisms from contaminated sites. Filamentous fungi can bioremediate xenobiotics thanks to their efficient enzymatic machinery. However, most studies on the degradation of 2,4-D have been carried out with bacteria, and little is known about whether it can be efficiently biodegraded by fungi. In the environment, fungal strains and native microbiota may detoxify contaminants through mechanisms like biosorption, bioabsortion, biotransformation, and/or degradation. Whether these processes occur separately or simultaneously depends on the metabolic ability of the strains that conform the microbial community. Another important concern when attempting to introduce detoxifying microorganisms into a contaminated environment is the GRAS ("Generally Recognized As Safe") assessment or status. These are studies that help predict a biodegrading microorganism's pathogenicity, toxicity, and infectivity before in situ application. This application, moreover, is regulated by different legal frameworks. The present review aims to outline the main aspects of 2,4-D degradation by fungi, and to summarize the current state of research on the topic in Argentina.

Influence of a glyphosate-based herbicide on growth parameters and aflatoxin B1 production by Aspergillus section Flavi on maize grains.

Glyphosate-based herbicides (GBH) are the main pesticides applied worldwide on maize production. Glyphosate-resistant weeds led to the repeated application of high doses of the pesticide. In addition to environmental conditions, the presence of GBH affects the development of Aspergillus species and aflatoxin B1 (AFB1) production under in vitro conditions. The aim of this work was to evaluate the influence of a commercial GBH on growth and AFB1 production by Aspergillus flavus and Aspergillus parasiticus strains under different water activity (aW) conditions. The following concentrations of active ingredient glyphosate were evaluated: 20, 50, 200 and 500mM. The lag phase prior to growth and growth rate did not change at 20 and 50mM (that is, at field recommended doses) at 0.98 and 0.95 aW; however, at increasing GBH concentrations, between 200 and 500mM, the growth rate decreased at all aW conditions. In general, as the GBH concentration increased, AFB1 production decreased. However, a significant increase in toxin accumulation was found only at one of the aW conditions (0.95) at 21 days with 50mM of GBH in A. flavus and 20 and 50mM of GBH in A. parasiticus. These results show that, even though Aspergillus section Flavi growth did not increase, AFB1 production increased on maize grains at GBH concentrations similar to those of field recommended doses under favorable water availability and temperature conditions.

Effects of chlorpyrifos on growth and aflatoxin B1 production by Aspergillus section Flavi strains on maize-based medium and maize grains.

Chlorpyrifos is one of the most used insecticides in agro-ecosystems and is repeatedly applied due to the increase in pest resistance, which leads to environmental accumulation. The aim of this work was to evaluate the effect of chlorpyrifos on growth and aflatoxin B1 (AFB1) production by four Aspergillus section Flavi strains, under different water conditions-aW (0.93, 0.95 and 0.98)-on maize-based medium (MMEA) and maize grains supplied with 0.06 to 1.4 mmol/L of chlorpyrifos. MMEA plates were incubated at 18, 28, and 37 °C and plates with maize grains at 25 °C during 21 days. Chlorpyrifos stimulated the growth and AFB1 production of non-target organisms, such as Aspergillus section Flavi strains, both at low (0.06 mmol/L) and at high concentrations (1.4 mmol/L) on MMEA and maize grains. Stimulation occurred over a wide range of temperature and aw conditions. The toxin concentration produced by the two strains on MMEA at 18 °C increased when the concentration of chlorpyrifos also increased, being most significant at 0.6 mmol/L. In conclusion, the presence of chlorpyrifos should be considered as a factor, together with environmental conditions, for the development of effective production practices of maize grains, in order to avoid fungal growth and AFB1 production, to prevent both economic losses and risks to human and animal health.

Isolation of culturable mycota from Argentinean soils exposed or not-exposed to pesticides and determination of glyphosate tolerance of fungal species in media supplied with the herbicide / Aislamiento de la microbiota cultivable de suelos argentinos expuestos y no expuestos a plaguicidas y determinación de la tolerancia a glifosato de especies fúngicas en medios suplementados con el herbicida

Abstract The current agricultural system has led to the development of glyphosate (GP)-resistant weeds, causing an increase in GP doses and applications. Native mycota of pesticide-contaminated sites are the major source of pesticide-degrading microorganisms. The aims of the present study were to isolate the GP-tolerant culturable mycota in two soils with different pesticide exposure from Córdoba, Argentina, and to evaluate the growth parameters in native fungal isolates in the presence of GP and the effective dose that caused 50% growth reduction. The results showed that the genera Fusarium, Aspergillus, Mucor, Penicil-lium and Sterilia were the prevalent fungi isolated from soils both exposed and not-exposed to pesticides. The highest value (>100 mM) of effective concentration of herbicide that caused 50% growth inhibition (EC50), was found for Trichoderma isolates. Sterilia spp. had EC50 val-ues of 100mM, while Aspergillus spp. and Mucor had EC50 values between 50 and 100mM. The growth rate evaluation varied according to the isolates and GP concentrations. The data showed that all Aspergillus spp., Trichoderma spp., Mucor and three Sterilia spp. had the best growth performance in media supplied with GP after a variable acclimation period. This study provides valuable data for further studies that would allow to know the metabolic capacity of these fungal species that can be potential candidates for GP removal from contaminated environments.

Resumen El sistema agrícola actual condujo ai desarrollo de malezas resistentes a glifos-ato (GP), lo que deriva en el aumento de las dosis y el número de aplicaciones requeridas. La microbiota nativa de sitios contaminados con plaguicidas constituye la fuente principal de microorganismos capaces de degradar este herbicida. Los objetivos del presente estudio fueron aislar la microbiota cultivable tolerante a GP a partir de dos suelos agrícolas con diferente grado de exposición a plaguicidas, ambos de la provincia de Córdoba, Argentina, y evaluar en los hongos nativos aislados los parámetros de crecimiento en presencia de GP y la dosis efectiva que produce un 50% de reducción del crecimiento (EC50). Los géneros Fusarium, Aspergillus, Mucor, Penicillium y Sterilia fueron los de mayor prevalencia, tanto en suelos expuestos como no expuestos a plaguicidas. La mayor EC50 hallada (> 100 mM) correspondió a dos aislamientos de Trichoderma. En dos aislamientos de Sterilia, los valores de EC50 fueron de 100 mM, mientras que en los dos de Aspergillus y en uno de Mucor fueron de entre 50 y 100 mM. La velocidad de crecimiento varió según la concentración de GP empleada y el aislamiento considerado. Todos los aislamientos de Aspergillus, Trichoderma y Mucor, y tres aislamientos de Sterilia mostraron el mejor rendimiento en cuanto al crecimiento en los medios con GP después de un período de aclimatación variable. Este trabajo proporciona datos preliminares valiosos referidos a la capacidad metabólica de estos aislados y su potencial para remover GP en ambientes contaminados.

Herbicides based on 2,4-D: its behavior in agricultural environments and microbial biodegradation aspects. A review.

One of the main herbicides used in the agricultural environments is 2,4-dichlorophenoxyacetic acid (2,4-D). It is a synthetic plant hormone auxin employed in many crops including rice, wheat, sorghum, sugar cane, and corn to control wide leaf weeds. The indiscriminate use of pesticides can produce numerous damages to the environment. Therefore, this review has the objective to provide an overview on the main characteristics of the herbicides based on 2,4-D, mostly on the role of microorganisms in its degradation and its main degradation metabolite, 2,4- dichlorophenol (2,4-DCP). The remediation processes carried out by microorganisms are advantageous to avoid the pollution of the environment as well as to safeguard the population health.

Isolation of culturable mycota from Argentinean soils exposed or not-exposed to pesticides and determination of glyphosate tolerance of fungal species in media supplied with the herbicide.

The current agricultural system has led to the development of glyphosate (GP)-resistant weeds, causing an increase in GP doses and applications. Native mycota of pesticide-contaminated sites are the major source of pesticide-degrading microorganisms. The aims of the present study were to isolate the GP-tolerant culturable mycota in two soils with different pesticide exposure from Córdoba, Argentina, and to evaluate the growth parameters in native fungal isolates in the presence of GP and the effective dose that caused 50% growth reduction. The results showed that the genera Fusarium, Aspergillus, Mucor, Penicillium and Sterilia were the prevalent fungi isolated from soils both exposed and not-exposed to pesticides. The highest value (>100mM) of effective concentration of herbicide that caused 50% growth inhibition (EC50), was found for Trichoderma isolates. Sterilia spp. had EC50 values of 100mM, while Aspergillus spp. and Mucor had EC50 values between 50 and 100mM. The growth rate evaluation varied according to the isolates and GP concentrations. The data showed that all Aspergillus spp., Trichoderma spp., Mucor and three Sterilia spp. had the best growth performance in media supplied with GP after a variable acclimation period. This study provides valuable data for further studies that would allow to know the metabolic capacity of these fungal species that can be potential candidates for GP removal from contaminated environments.

Effect of in vitro glyphosate on Fusarium spp. growth and disease severity in maize.

BACKGROUND: Glyphosate (GP) is one of the main pesticides used for maize production. Fusarium sp. is a fungal genus with several phytopathogenic species and toxigenic features. In this study, the culturable soil mycota was evaluated using the surface-spray method. The effect of GP on the growth parameters (growth rate and lag phase) of Fusarium spp. was also tested on solid media conditioned with different water activities. Finally, the GP effect on disease severity caused by Fusarium sp. in maize seedlings was studied. RESULTS: The results showed that Fusarium species are frequently isolated from GP-exposed soils. The GP concentrations tested had a significant effect on F. graminearum, F. verticillioides and F. oxysporum growth parameters on solid media. The pathogenicity tests showed that the disease severity of the maize seedlings significantly increased with increasing GP concentrations. CONCLUSIONS: This study showed that Fusarium species are frequently isolated from pesticide-exposed soils and the GP concentrations tested had a significant effect both on growth parameters and disease severity in maize. This study provides an approach to the effect of GP on Fusarium sp. growth and pathogenicity that reinforces the importance of evaluating all the factors that could affect feed and food production. © 2019 Society of Chemical Industry.

Desarrollo y capacidad de remoción de los pesticidas atrazina, clorpirifós y endosulfán por parte de cepas de Aspergillus sección Flavi no toxigénicas / Development and removal ability of non-toxigenic Aspergillus section Flavi in presence of atrazine, chlorpyrifos and endosulfan

This study evaluated the in vitro effect of three concentrations of atrazine, chlorpyrifos and endosulfan on the growth parameters of four non-toxigenic Aspergillus section Flavi strains. The ability of the strains to remove these pesticides in a synthetic medium was also determined. Growth parameters were measured on soil extract solid medium supplied with 5,10 and 20mg/l of each pesticide, and conditioned to -0.70, -2.78, -7.06 and -10.0 water potential (MPa). Removal assays were performed in Czapek Doc medium (CZD) supplied with 20mg/l of each pesticide under optimal environmental conditions (-2.78 of MPa and 25 °C). The residual levels of each pesticide were detected by the reversed-phase HPLC/fluorescence detection system. The lag phases of the strains significantly decreased in the presence of the pesticides with respect to the control media. This result indicates a fast adaptation to the conditions assayed. Similarly, the mycelial growth rates in the different treatments increased depending on pesticide concentrations. Aspergillus oryzae AM 1 and AM 2 strains showed high percentages of atrazine degradation (above 90%), followed by endosulfan (56 and 76%) and chlorpyrifos (50 and 73%) after 30 days of incubation. A significant (p <0.001) correlation (r = 0.974) between removal percentages and growth rate was found. This study shows that non-toxigenic Aspergillus section Flavi strains from agricultural soils are able to effectively grow in the presence of high concentrations of atrazine, chlorpyrifos and endosulfan under a wide range of MPa conditions. Moreover, these strains have the ability to remove high levels of these pesticides in vitro in a short time.

En este estudio se evaluó los efectos in vitro de 3 concentraciones de atrazina, clorpirifós y endosulfán sobre los parámetros de crecimiento de 4 cepas no toxigénicas de Aspergillus sección Flavi. También se evaluó la capacidad de las cepas de remover los pesticidas. Los parámetros de crecimiento se ensayaron en medio agar extracto de suelo suplementado con 5, 10 y 20mg/l de cada pesticida y acondicionado a -0.70, -2.78, -7.06 y -10.0 de potencial de agua (MPa). Los ensayos de remoción se realizaron en medio Czapek Dox con 20mg/l de cada pesticida bajo condiciones óptimas de crecimiento (-2.78 de MPa y 25 °C). Los niveles residuales de atrazina, clorpirifós y endosulfán se detectaron en un sistema HPLC con detección por fluorescencia. La fase de latencia de las cepas disminuyó significantemente en presencia de los pesticidas, indicando una rápida adaptación a dichas condiciones. La velocidad de crecimiento se incrementó considerablemente dependiendo de la concentración de pesticida. Las cepas Aspergillus oryzae AM1 y AM2 mostraron porcentajes elevados de degradación de atrazina (aproximadamente el 90%), seguidos por endosulfán (56 y 76%) y clorpirifós (50 y 73%). Se observó una correlación (r = 0.974) significante (p <0.001) entre el porcentaje de pesticida removido y la velocidad de crecimiento. Este estudio muestra que cepas no-toxigénicas de Aspergillus sección Flavi aisladas de suelos agrícolas desarrollan eficientemente en presencia de altas concentraciones de atrazina, clorpirifós y endosulfán en un amplio rango de MPa. Además, presentan capacidad de remover in vitro altos niveles de pesticidas en corto tiempo.

Development and removal ability of non-toxigenic Aspergillus section Flavi in presence of atrazine, chlorpyrifos and endosulfan.

This study evaluated the in vitro effect of three concentrations of atrazine, chlorpyrifos and endosulfan on the growth parameters of four non-toxigenic Aspergillus section Flavi strains. The ability of the strains to remove these pesticides in a synthetic medium was also determined. Growth parameters were measured on soil extract solid medium supplied with 5, 10 and 20mg/l of each pesticide, and conditioned to -0.70, -2.78, -7.06 and -10.0 water potential (MPa). Removal assays were performed in Czapek Doc medium (CZD) supplied with 20mg/l of each pesticide under optimal environmental conditions (-2.78 of MPa and 25°C). The residual levels of each pesticide were detected by the reversed-phase HPLC/fluorescence detection system. The lag phases of the strains significantly decreased in the presence of the pesticides with respect to the control media. This result indicates a fast adaptation to the conditions assayed. Similarly, the mycelial growth rates in the different treatments increased depending on pesticide concentrations. Aspergillus oryzae AM 1 and AM 2 strains showed high percentages of atrazine degradation (above 90%), followed by endosulfan (56 and 76%) and chlorpyrifos (50 and 73%) after 30 days of incubation. A significant (p<0.001) correlation (r=0.974) between removal percentages and growth rate was found. This study shows that non-toxigenic Aspergillus section Flavi strains from agricultural soils are able to effectively grow in the presence of high concentrations of atrazine, chlorpyrifos and endosulfan under a wide range of MPa conditions. Moreover, these strains have the ability to remove high levels of these pesticides in vitro in a short time.