Ciencias Agropecuarias
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Item Aislamiento, caracterización e identificación de bacterias con capacidad fijadora de nitrógeno atmosférico asociadas con la rizosfera de Solanum tuberosum L. Var. Superchola(2024-02) Muñoz Muñoz, Kevin Alexis; Leiva Mora, MichelThe present research was developed with the purpose of isolating, characterizing and identifying in vitro atmospheric nitrogen-fixing bacteria associated with the rhizosphere of S. tuberosum var. Superchola; as well as to determine their effect on the growth of potato vitroplants in greenhouses. Isolates were obtained from four localities in the province of Tungurahua, which were culturally and morphologically characterized. The in vitro atmospheric nitrogen fixation capacity was determined using modified Jensen culture medium, and molecular identification was performed by PCR amplification of 16S rRNA genes. Finally, the effect of the bacterial isolates on the growth of potato vitroplants in greenhouses was determined. The results showed that in the canton of Píllaro there was the highest quantity of CFU. g-1 of soil (9.08 x 109), in the cultural characterization 87% had smooth texture, 42% flat surface, 82% smooth consistency, 42% circular shape, 84% entire margin and 95% showed brightness. In the morphological characterization of the isolates, 84% were Gram (-) bacteria and 95% were bacilli. The bacterial isolate with the highest atmospheric nitrogen-fixing potential was CC-FCAGP-BFN1 and the one with the lowest potential was CC-FCAGP-BFN15. PCR amplification of 16S rRNA genes identified the strains Pseudomonas sp. (CC-FCAGP-BFN1, CC-FCAGP-BFN16), CC-FCAGP-BFN14) and Paraburkholderia dipogonis (CC-FCAGP-BFN24), which showed a high capacity for atmospheric nitrogen fixation. Likewise, the bacterial strain CC-FCAGP-BFN1 (Pseudomonas sp.) was the only one that increased leaf area, fresh and dry mass and leaf area index of S. tuberosum vitroplants. Based on the above results, four bacterial strains were identified from the rhizosphere of S. tuberosum, one of which was the most promising for favoring vegetative growth due to its high capacity to fix atmospheric nitrogen.Item Evaluación de antibiorresistencia en bacterias grampositivas aisladas en carne de pollo que se expende en el cantón Ambato, provincia de Tungurahua(2024-02) Velastegui Viteri, Cristian David; Cruz Quintana, Sandra MargaritaThe present investigation was carried out with the main purpose of evaluating the antibacterial resistance in Gram positive bacteria (Vagococcus lutrae, Mammaliicoccus sciuri and Kurthia gibsonii) isolated in chicken meat sold in Ambato Canton, province of Tungurahua, because bacterial resistance to antibiotics is a specific aspect of the natural evolution of bacteria, The study was carried out with strains of (Vagococcus lutrae, Mammaliicococcus sciuri and Kurthia gibsonii) that were isolated and molecularly identified from chicken meat sold in the canton of Ambato from previous investigations, which are kept in the Laboratory of Microbiology and Molecular Biology of the Faculty of Agricultural Sciences of the Technical University of Ambato. It was found that there is the presence of antibioresistance in gram-positive bacteria, specifically in strains of Vagococcus lutrae, Mammaliicoccus sciuri and Kurthia gibsonii, isolated from samples of chicken meat marketed in the Ambato Canton, province of Tungurahua. Likewise, the results obtained by the agar diffusion method revealed significant patterns of antibiotic resistance in the Gram-positive bacteria studied. Resistance profiles against various antibiotics used in both Veterinary and Human Medicine were identified, highlighting the importance of addressing resistance in an interdisciplinary context and the analysis of virulence factors in strains of Vagococcus lutrae, Mammaliicococcus sciuri and Kurthia gibsonii provided valuable information on the possible risks associated with these bacteria in chicken meat. Keywords: EVALUATION, ANTIBIORRESISTANCE, BACTERIA, GRAM POSITIVE, CHICKEN MEAT, AMBATO, TUNGURAHUA.Item Evaluación de la actividad antimicrobiana del aceite esencial microencapsulado de Hierba Luisa (Cymbopogon citratus) sobre bacterias Gram positivas aisladas de piel de conejo (Oryctolagus cuniculus)(2024-02) Jaramillo Panimboza, Katherine Salomé; Cruz Quintana, Sandra MargaritaThe objective of the research was to evaluate the antimicrobial activity of the microencapsulated essential oil of Lemongrass (Cymbopogon citratus) on Grampositive bacteria isolated from rabbit skin (Oryctolagus cuniculus), two Gram-positive bacteria (Paenibacillus profundus and Staphylococcus succinus) were molecularly identified using a 16s marker, the agar diffusion method in wells to measure antimicrobial activity, determined that the two bacterial strains did not form halos of inhibition, the minimum inhibitory concentration (MIC) in liquid medium was determined at 5% for the P. profundus strain with an optical density of 0.251, with respect to the S. succinus strain it was established that at 25% concentration of the oil there was bacterial growth at an optical density of 0.487, the colony forming units (CFU) for the S. succinus strain were determined at 15% of the oil concentration, with an average range of 2.00 CFU, being the best treatment, while for the P. profundus bacteria it was established from 15%, 20% and 25%, as it does not exist in any of them. the growth of UFC.Item Evaluación de la capacidad de fijación del nitrógeno atmosférico de las bacterias asociadas con la rizosfera de plantas de Solanum tuberosum L. Var. Superchola en la provincia de Tungurahua(2023-09) Altamirano Diaz, Vanessa Carolina; Leiva Mora, MichelThe present study was developed with the purpose of isolating, quantifying, characterizing, evaluating and conserving free-living bacteria associated with the potato rhizosphere with atmospheric nitrogen fixation capacity, from soil samples from four localities in the province of Tungurahua. First, soil samples were collected from the potato crop and placed in plastic containers of 6 L capacity, where tubers of the Superchola variety were planted. After 60 days, samples of rhizospheric soil (area near the roots) were extracted. For isolation, the pour plate method was used in Jensen culture medium free of nitrogen sources. To quantify viable bacteria per gram of soil from each locality, serial solutions were prepared where 100 µl were deposited in solid medium, incubated for 72 hours at 30 ºC in inverted position and the plate count was performed. The locality of Santa Rita in the canton of Pillaro had the highest number of CFU/g of soil. A total of 72 isolates were obtained from the potato rhizosphere, of which only 38 isolates produced a positive reaction with the color change of the medium modified with bromothymol blue from green to blue, these were subjected to morphological and cultural characterization. In relation to morphology, 84% were predominantly Gram (-) bacteria, bacilli (95%) and their grouping was mostly in the form of diplobacilli (37%). In the cultural characterization, the following shapes predominated: circular (42%), entire margin (84%), smooth texture (87%), flat surface (42%), smooth consistency (82%) and 95% showed brightness. The bacterial isolate with the greatest capacity to fix atmospheric nitrogen was (T1-FCAGP-BFN1) from the El Placer sector of the Quero canton, which had no statistically significant difference with respect to isolates T3-FCAGP-BFN14, T3-FCAGP-BFN16 and T4- FCAGP-BFN24. The purified isolates were deposited in eppendorf tubes with 15% glycerol and BHI liquid medium + 20% glycerol and stored at -20 ºC. The bacterial isolates with the highest nitrogen-fixing capacity obtained could be used to determine their plant growth-promoting activity in greenhouses prior to their possible use as biofertilizers under field conditions.Item Identificación molecular de aislados bacterianos contaminantes de carne de pollo (Gallus gallus) expendida en puntos de venta del cantón Ambato(2023-03) Escobar Aguilar, Solange Belén; Cruz Quintana, SandraFood of animal origin is exposed to several contamination factors in the production chain. Based on this argument, the objective of this research was the molecular identification of bacterial isolates contaminating chicken meat (Gallus gallus) sold in points of sale in the Ambato canton. The methodology started with 130 bacterial isolates, continued with the extraction of DNA, evaluating its quality by means of spectrophotometry with absorbance of 260/280 nm, determining its purity. For PCR using the 16S ribosomal DNA region, the universal primers 27F and 1492 R were used, and then electrophoresis was performed. The size of the bands was 1 500 base pairs (bp), the samples were sequenced and the identity of the processed bacteria was obtained, which were: Proteus mirabilis (36.09%), Escherichia coli (30.83%), Shigella sonnei (7.52%), Shigella flexneri (3.76%), Shigella dysenteriae (1.5%), Shigella sp. (0. 75%), Salmonella enterica (1.5%), Escherichia fergusonni (5.26%), Mammaliicoccus sciuri (2.26%), Enterobacter cloacae (1.5%), Proteus vulgaris (1.5%), Pseudescherichia vulneris (1.5%), Kurthia gibsonii (1.5%), Proteus sp. (0.75%), Comamonas kerstersii (0.75%), Cronobacter sakazakii (0.75%), Vagococcus lutrae (0.75%), Escherichia marmotae (0.75%) and Klebsiella variicola (0.75%). Finally, to compare the sequences deposited in the GenBank database, the content of Guanines + Cytokines, which are related to pathogenic islands, was taken into account; the bacterium with the highest content was Cronobacter sakazakii with 59.6%. In conclusion, the raw chicken meat sold in this territory contains a high index of microbiological contamination, demonstrating that one of the causes for the development of foodborne diseases (FBD) is the inadequate handling of the product, thus representing a serious problem for public health.Item Aislamiento de bacterias asociadas con la rizosfera de Solanum tuberosum L. var. Superchola en dos localidades de la provincia de Tungurahua(2023-03) Altamirano Mayorga, Christian Alejandro; Leiva Mora, MichelThe purpose of this research was to isolate and characterize the cultivable bacteria present in the roots (rhizobacteria) of the potato of the Superchola variety in two locations in the province of Tungurahua. In the first phase, the examination of soils where potatoes are grown was carried out, to later place them in plastic bottles with a capacity of 6000 ml; where the tubers of the Superchola variety were planted. In this way, 50 days elapsed, root samples were taken at each of the depths (8, 16 and 24 cm) to carry out the isolation by 3 methods (Inclusion of 10 mm long root fragments, Washing planting and by root dissemination, and Maceration of root fragments in NaCl saline buffer and dissemination), for which the nutrient agar culture medium was obtained. It was possible to obtain the number of 82 isolated bacteria obtained in their predominant cultural characterization in 84% with a circular shape, 85% with an entire border, 93% colonies with shine, 67% with a flat elevation and 88% with a smooth texture. In relation to its morphological characterization, Gram (+) bacteria predominated in 58%, bacilli 97%. To determine the amount of CFU/g of root, serial solutions of each location and depth were made, leaving them to incubate for 48 hours at 28°C and thus proceed to count. In order to measure the diversity of cultivable bacteria in the soil, 250 g of the sample from each location were taken, which were sent to the IDgen laboratory, where the metagenomic analysis was carried out, in which the report mentions the prevalence of 7856 cultivable bacteria in the soil. the town of Santa Rita in the canton of Píllaro and 1307 in the town of Llangahua in the canton of Ambato. Based on the results obtained, it could be said that in the Santa Rita locality of the Píllaro canton there is a greater bacterial biodiversity. It would be very interesting if each of the isolates obtained is used for research that seeks the creation of bio-inputs for this type of staple crop in the diet of the population.Item Evaluación de la capacidad de solubilización de fosfatos por bacterias asociadas a la rizosfera de papa Solanum tuberosum L. Var. Superchola(2023-03) Mendoza Mina, Hugo Toniño; Leiva Mora, MichelThe objectives of this research were to evaluate, select, characterize and conserve the bacterial isolates associated with the potato rhizosphere with the highest phosphate solubilization capacity from four potato-producing localities in the province of Tungurahua, starting with the planting of potato tubers of the Superchola variety in 6 L pots, which after 45 days from sprouting, the bacteria that presented phosphate solubilization halos were isolated, for which the Pikovskaya Agar culture medium was prepared. A completely randomized design was used, the bacterial isolates from the locality with the highest average ranges were from the locality of Pinguilí in the Mocha canton. Of the 40 bacterial isolates, the one with the lowest capacity to solubilize phosphates was isolate CC-FCAGP-BSF10, although it had no significant statistical difference with respect to isolates CC-FCAGP-BSF12, CC-FCAGP-BSF19, CC-FCAGP-BSF13, CC-FCAGP-BSF15, CC-FCAGPBSF14 and CC-FCAGP-BSF31. Of the 40 culturally characterized isolates, 28 % were dominated by umbelliform elevation, 62 % by circular colony forms, 85 % by entire edges, 92 % by shiny, 92 % by smooth texture and 80 % by colonies of smooth consistency. In morphology, bacilli predominated with 50 %, in grouping, diplobacilli predominated with 45 %, and in Gram reaction, Gram (-) bacteria predominated with 83 %. For the quantification of bacteria per gram of soil of each locality, serial dissolutions were made after 48 hours of incubation at 28ºC, proceeding to the count of CFU.g per soil of each locality, giving as a result that the locality of Santa Rita del Cantón Píllaro was the one that reached the highest average range. For the preservation of the bacterial isolates with the highest phosphate solubilization capacity, 1.5 ml eppendorf tubes were used with two variants: 25% glycerol or 20% glycerol plus brain heart infusion culture medium (BHI). According to the results obtained, the isolates with the highest phosphate solubilization capacity were found in the locality of Pinguilí in Mocha county, which are promising candidates for biofertilizers, while the isolates with the lowest solubilization capacity were found in the locality of El Placer in Quero county, which at the same time was the locality with the lowest number of bacteria per gram of soil, demonstrating the reduced bacterial biodiversity of this locality.Item Compatibilidad y tiempo de sobrevivencia de tres bacterias benéficas de uso agrícola (Bacillus thurigiensis, Bacillus subtilis, Bacillus laterosporus), en bioles(2021-09) Rodríguez Benavides, Pablo Aníbal; Vásquez Freytez, Carlos LuisAt present, the contamination and loss of macro and micro fauna and flora due to the excessive use of pesticides, allows the attack of pests and diseases in crops to be more aggressive and difficult to control. The soil feeds living beings in which millions of microorganisms coexist, forming a total balance of survival. Microorganisms allow plant nutrition, control pests and diseases in crops. In this scientific proposal, the compatibility and survival time of three bacteria of the Bacillus genus, for agricultural use, were evaluated for the improvement of bioles. In each treatment the number of CFU (colony forming units) was evaluated at 30, 60 and 90 days after inoculation in a biol substrate, the treatments were compared with a control where the bacteria were not inoculated. According to the results, the lowest population values were observed 1 month after inoculation of the biol with the different species of Bacillus, in which no effect of the species of bacteria or the applied dose was observed, observing that the number of CFU varied from 188.4 in B. subtilis up to 1186.1 in B. thuringiensis. The evaluations at the second month showed that there was an interaction effect between the bacteria and the dose used, noting that the greatest increase in the number of CFU occurred when B. laterousporus was used at 150 mL / 20L of biol in which it reached 2824.0 CFU (table 8). On the other hand, the following combinations resulted in the lowest populations were B. laterosusporus at 50 mL, B. thuringiensis at the 100 mL dose and B. subtilis at 100 and 150 mL doses. The rest of the treatments showed intermediate values that ranged between 1114.6 and 2215.3 CFU when B. thuringiensis was used at doses of 50 and 150 mL, respectively.Item Persistencia de bacterias entéricas resistentes y genes de resistencia a antibióticos en sustratos suplementados con gallinaza comercial y de compostaje tradicional(2021-01) Bohórquez Pucha, Jandry David; Calero Cáceres, William RicardoThe application of animal manure (fresh or composted) represents a common practice. This practice favors the increasing of organic matter into soils at reduced costs. The efficient use of the animal residues supporting the recycling of organic matter and nutrients. Compost practices transforms efficiently and safely animal manure into materials suitable for use into agricultural soils. However, the application of traditional practices to compost fertilizers, consisting of simple drying of the animal manure and later applying it directly to the soil is common. The high levels of enteric bacteria present in these matrices may represent a pathway for the spread of pathogenic organisms, which can potentially integrate within the food chain. For this reason, the persistence of enteric bacteria (heterotrophic bacteria, E. coli, coliforms) resistant to antibiotics and the persistence of antibiotic resistance genes were evaluated in soils supplemented with two types of fertilizer (compost of animal origin traditionally composted and compost of animal origin processed by the industry) during 111 days; determining that the application of these fertilizers notably increases the densities of resistant enteric bacteria (ARBs) and antibiotic resistance genes (ARGs) in soils. These pollutants have a high persistence over time, which suggests that the application of fertilizers of animal origin, especially when they do not receive appropriate treatment, may represent a pathway for the spread of antibiotic resistance determinants throughout the chain. fooItem Efecto de la aplicación de gallinaza de compostaje tradicional y comercial sobre las densidades de bacterias resistentes y genes de resistencia a antibióticos en cultivo de rábano (Raphanus sativus) y lechuga (Lactuca sativa)(2020-09) Cruz Morales, Carla Marisol; Calero Cáceres, William RicardoPoultry manure is traditionally used as a good source of nutrients and organic matter in agricultural soils, especially in organic-farming systems. However, these materials can cause adverse effects on general soil conditions, since they contain high densities of potentially pathogenic enteric bacteria; such as antibiotic resistant bacteria (ARBs) and their corresponding antibiotic resistance genes (ARGs). The use of these materials may represent a probable route of dissemination of pathogenic microorganisms and their determinants through the food chain. This thesis is part of the project "EVALUACIÓN DE LA DISEMINACIÓN AMBIENTAL DE GENES DE RESISTENCIA A ANTIBIÓTICOS POR MEDIO DEL ESTIÉRCOL DE GALLINA" (HCU 1752-CU-P-2019). The aim of this thesis is the evaluation of the effect of the addition of traditional and commercial composting on the densities of ARBs and ARGs in vegetables (lettuce and radish). The virulence and resistance mechanisms of the detected E. coli isolates were evaluated. The results suggest that traditional composting chicken manure influences to a greater extent the spread of ARBs and ARGs in relation to commercial chicken manure. The E. coli isolates presented an antibiotic multiresistance profile. The transconjugation experiments suggest the mechanisms of antibiotic resistance of these bacteria were found encoded in chromosomes and not in plasmids. On the other hand, the qPCR technique was used to detect ARGs, which showed very high levels of detection for sul1 and blaTEM and low levels of detection to qnrS, tetW, ermB and 16S rDNA. The dissemination of ARBs and ARGs from traditional and commercial poultry manure to food is plausible, representing this a potential route of dissemination of antibiotic resistance determinants through the food chain.