FRRE - Producción de Investigación
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Item Analysis of herbicide biodegradation by native bacteria isolated from rice crop soils in Chaco, Argentina(Sociedad Argentina de Microbiología General, 2024-12-10) Cuadra, Pablo Nicolás; Farías, Alejandro Rubén; Jorge, Nelly Lidia; Vullo, Diana LiaIn Argentina, weed management in rice and other crops is often done with herbicides that persist in the soil for a long time. The incorrect and indiscriminate use of these agrochemicals has caused environmental problems, such as soil, groundwater and surface water contamination. Clomazone (CLM), an herbicide from the chemical group of oxazolidinones, is a selective pre- and post-emergence herbicide indicated for application in rice. Imazapyr and imazapic (IMR+IMC) are two herbicides that belong to the group of imidazolinones which interfere weed growth by inhibiting the action of the plant enzyme acetohydroxyacid synthase (AHAS). The objective of this work was to analyze the growth kinetics (GK) of bacterial strains isolated from rice crop soils with CLM and IMR+IMC as the only carbon source, and to analyze their biodegradation over time.Item Growth kinetics of soil bacteria in presence of pesticides applied for rice crop production(2023-10-05) Cuadra, Pablo Nicolás; Farías, Alejandro Rubén; Utgés, Enid Marta; Jorge, Nelly Lidia; Vullo, Diana LiaThe expansion of agricultural activity promoted the use of agrochemicals, whose permanence in the soil can cause serious contamination problems. Its infiltration into groundwater or runoff into surface watercourses, as well as their deposition in trophic chains, imply a latent risk to the balance of ecosystems. The present work objective is to analyze the growth kinetics of bacteria isolated from soils destined for rice cultivation in Las Palmas, Chaco, Argentina, in the presence of glyphosate, imazapic-imazapyr and clomazone, pesticides of current application. Two of 24 isolated bacteria named H5 and I2 were specially selected for this study because of their resistant to high agrochemical concentrations. Growth kinetics tests were carried out applying two methods to monitor biomass increase. One of these methods was based on biomass determination by dry weight (BPS) and the other by colony forming units/ml (CFU/ml) count using the drop technique (RMG). Initial inoculums were prepared in minimal saline medium (M9) with glucose 0,5%p/V and incubated at 30°C at 200 or 300 rpm for 24 h. Then, 5 ml of each were transferred to 50 ml of M9 with 1% V/V of the mixture of agrochemicals as the only carbon source to evaluate growth kinetics under these restrictive conditions. The formation of bacterial aggregates at the lowest agitation level was observed, interfering with biomass determinations. For this reason, the subsequent tests were carried out at 300 rpm to prevent the floc formation. To apply the BPS method, 1 ml culture samples were centrifuged 30 minutes, and the pellet obtained was dried for 10 h at 60°C to be subsequently weighed. For the RMG method, culture samples were taken and 10 µl drops of serial dilutions in NaCl 150 mM, were seeded in triplicates in Plate Count Agar (PCA). The growth kinetics was also analyzed by RMG with glucose as sole carbon source. The emulsification of clomazone formulation derived in turbidity in culture medium, generating interferences in the biomass estimation by the BPS method. Therefore, the only way to evaluate kinetic parameters was by the CFU/ml counting method by RMG at 300 rpm. In this way, growth curves for the two tested bacteria revealed generation times (tg) of 1.48 h and 7.46 h with division rates () of 0.68 h-1 and 0.15 h-1 for I2 and H5 respectively. Results obtained with glucose showed higher division rates and shorter generation times (I2=1,4 h-1,H5= 0,48 h-1, tgI2=0,73 h, tgH5=2.34 h) demonstrating a slowdown in bacterial growth by pesticide formulation consume. Further studies will be focused on the identification of degradation products to proceed with the application of mixed cultures in rice crop soil bioremediation microcosms experiments.Item Selection of native bacteria from rice soils resistant to glyphosate, clomazone and Kifix, to be used as biodegrading agents(2022-10-28) Cuadra, Pablo Nicolás; Fontana, Gimena Lilián; Farías, Alejandro Rubén; Jorge, Nelly Lidia; Vullo, Diana LiaRice is the main crop worldwide, covering about 11% of the planet’s arable land and constitutes a strategic component of the diet adopted for more than half of the world population. In Argentina, 3% of its production is in the Province of Chaco. Glyphosate, clomazone and kifix, are herbicides commonly used in rice crops for weed control, generating alterations in the soil ecosystem, and affecting the trophic chain. In this province, several studies reported poisoning cases in humans by herbicide applications, particularly in La Leonesa. Consequently, local producers requested assistance to find a prompt solution for this environmental concern. As demonstrated, the ability of certain soil bacteria to degrade these types of pollutants ensures an onsite detoxification process. For this reason, the objective of this work was to isolate native bacteria from soils of rice crops, resistant to a mixture of agrochemicals composed by the commercial formulates of glyphosate, clomazone and kifix, to be applied in biodegradation tests. The soils samples were taken from 3 areas of a field intended for rice cultivation, located in La Leonesa: 1. Virgin area without registered pesticide applications (ZA), 2. Area without applications for a decade (ZB), and 3. Crop area with continuous application of agrochemicals (ZC). As a first approach, counts of cultivable microorganisms were carried out in a traditional nutrient medium (PCA) and an oligotrophic medium (AS= soil agar) with and without pesticides (CP and SP respectively). Differences were detected both in number and in the diversity according to the sampling area, the composition of the medium and the presence of pesticides. In parallel, mixed cultures were obtained from soil suspensions in a saline broth (M9) with 0.5% m/v glucose and 1% v/v of the same herbicides, obtaining new isolates in M9 agar (AM9). As a result, 24 strains were selected and purified from AS-CP, PCA-CP and AM9. Toxicity was evaluated by exposing these isolates to increasing concentrations of herbicides in agar cultures, registering a greater growth inhibition in AS-CP with respect to PCA and AM9, probably related to pesticide interactions with medium components lowering the bioavailability. From these tests, 16 strains were selected according to their resistance to the highest concentrations of the mixture (from 0.25% in AS-CP to 15% in PCA-CP). Finally, resistance assays were performed in M9 broth supplemented with increasing concentrations of herbicides (1-20% v/v), detecting 9 strains capable of developing up to 15% v/v. In further studies, pesticide biodegradation by-products will be screened in cultures supernatants. Regarding that the agrochemical applications in crops will eventually not be discontinued, this work represents the first step to develop an alternative based on native bacteria bioinoculants for the future quality recovery of Chaco rice production soils.Item Aislamiento de bacterias autóctonas de suelos de cultivos de arroz con aplicaciones periódicas de agroquímicos(2022-06) Cuadra, Pablo Nicolás; Fontana, Gimena Lilián; Farías, Alejandro Rubén; Jorge, Nelly Lidia; Vullo, Diana LiaLa expansión de la actividad agrícola ha impulsado el uso de agroquímicos, cuya permanencia en los suelos puede provocar serios problemas de contaminación. Su infiltración hacia las aguas subterráneas o escurrimientos hacia cursos de agua superficiales, como también la deposición de éstos en las cadenas tróficas implican un riesgo latente para el equilibrio de los ecosistemas. El presente trabajo posee como objetivos aislar bacterias autóctonas a partir de suelos agrícolas en presencia de una mezcla de agroquímicos (glifosato, clomazone e imazapir-imazapic) para utilizarlas potencialmente en ensayos de recuperación de suelos contaminados. Las muestras de suelo fueron tomadas de tres zonas de un campo destinado al cultivo de arroz: 1. zona virgen sin aplicaciones registradas de plaguicidas (ZA), 2. zona sin aplicaciones desde hace 1 década (ZB), y 3. zona de cultivos con aplicación continua de agroquímicos (ZC). Primeramente, para explorar la población microbiana cultivable resistente a los plaguicidas mencionados, se realizó un recuento en placa por siembra en superficie sobre dos medios de cultivo: medio nutritivo (Agar para recuento=APC) y medio oliogotrófico simulando el entorno natural (Agar Suelo=AS) tanto en presencia como en ausencia de agroquímicos (CP y SP respectivamente). Se obtuvieron diferencias tanto en los recuentos como en la diversidad de colonias acorde a la zona de muestreo, a la composición del medio y a la presencia de plaguicidas. Se observó una mayor disminución en UFC/g correspondiente a ZA en los medios suplementados con agroquímicos, consistente con sus antecedentes de no exposición, no así con ZB y ZC. Ante la diversidad de colonias desarrolladas en AS-CP y APC-CP; se aislaron y purificaron en tres pasos 15 cepas. Estos aislamientos serán utilizados en ensayos de biodegradación de plaguicidas para utilizarlas potencialmente en procesos de biorremediación a futuro.