Ciencias Agropecuarias

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    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, Michel
    The 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.
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    Aislamiento, caracterización cultural, morfológica, patogénica e identificación de genes AVR en Cladosporium fulvum Cooke.
    (2023-12) Sánchez Ortiz, Aldo Martin; Leiva Mora, Michel
    Gray mold caused by Cladosporium fulvum was described by Cooke in 1883, which develops in nightshades, specifically in tomato crops, causing damage from the youngest leaves to the top of the plant. The objective of this research was to "Isolate and characterize culturally, morphologically and pathogenically monosporic isolates of Cladosporium fulvum" obtained from signs of gray mold on Solanum lycopersicum leaves to identify the presence or absence of Avr genes in the cantons of the province of Tungurahua (Ambato, Baños, Cevallos, Mocha, Patate, Pelileo, Píllaro, Tisaleo). For isolation, the leaf printing method was applied in potato dextrose (PDA) culture medium with gentamicin sulfate, to describe the cultural and morphological characteristics, the microculture method was used accompanied by the observation of fungal structures under an optical microscope. of transmitted light. In the pathogenic characterization, the isolates of C. fulvum were activated The conidia were counted in the Neubauer chamber of the differential cultivars previously sown on substrates and the inoculum was applied with a manual sprayer. For amplification, DNA extraction and lyophilization of each of the isolates was carried out prior to development. of conventional PCR with two denaturations at 94°C with different cycles and an annealing at 55°C and 34 cycles with a final extension of 72°C. Obtaining that in the 8 cantons of the province of Tungurahua they all showed olive green colors on the obverse, while on the back it was black, with a superficial elevation, plush texture and irregular shape, lobed edges and in some isolates there was pigmentation and perspiration liquid, the mycelial growth in the colonies was obtained with a length of 5.93 µm, width 2.8 µm, hyphae 5.37 µm and conidiophores 32.97 µm average values. In the pathogenic characterization it was evident that the degree of affectation 1 represented between 1 to 5% of the surface of leaves with signs of C. fulvum, while 2 varied from 6 to 20% in the differential cultivars (Cf0-Cf2- Cf4- Cf5-Cf6), finally in the detection of the presence or absence of genes, the presence of the genes Avr2, Avr4e, Ecp2, Ecp4 and Ecp5 and total absence of the genes Avr4, Avr9 and Ecp1 were observed. Based on the results and the tests carried out, it was possible to create bases for the selection of resistance genes for the production of S. lycopersicum hybrids in the province of Tungurahua.
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    Aislamiento y caracterización del agente causal de pudriciones en el cultivo de Allium sativum L.
    (2023-09) Tituaña Cajahuishca, Mónica Tatiana; Guerrero Cando, David Aníbal
    Garlic (Allium sativum L.) is one of the crops with the smallest planting area in Ecuador due to its phytosanitary problems and the lack of quality seed bulbs. One of the factors that affects the production of this crop is the presence of diseases, among the most common is bulb rot. For this reason, the present investigation was proposed with the objective of identifying the causal agents that occur in the rotting of the garlic crop in the Pilahuín parish belonging to the Tungurahua province. Three isolates were obtained from infected bulb tissue, which were selected for cultural and morphological characterization. The results indicated that the causative agents of the most common rots were Fusarium oxysporum f.sp. cepae, Penicillium allii and Sclerotium cepivorum Berk. It is hoped that the results of these studies serve as a basis for the establishment of an integrated disease management program that leads to an increase in the garlic (Allium sativum L.) planting area in order to reach a supply in Ecuador.
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    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, Michel
    The 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.
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    Aislamiento y caracterización del agente causal del manchado de la hoja y necrosis del fruto de Capsicum annuum L. en cinco localidades de la Provincia de Tungurahua y una localidad de la provincia de Chimborazo
    (2023-03) López Villacis, María Liliana; Santana Mayorga, Rita Cumandá
    Leaf spotting and fruit necrosis caused by C. fulvum causes chlorotic spots on the upper side of the leaves that turn yellowish as the pathogen progresses and dark spots on the underside that turn olive green, causing partial or total loss of leaves and exposing the fruit to direct sunburn. The fruit shows tan to black lesions that, as the pathogen progresses, form a kind of olive-green velvety mass, causing great economic problems for growers. The objective of this research is to obtain monosporic isolates of the causal agent of leaf spot and fruit necrosis, the characterization of the colonies, the description of morphological characteristics, the inoculation of healthy leaves and fruits, where 106 solutions were made and finally the reisolation from inoculated leaves and fruits. Six monosporic isolates obtained from leaves and six monosporic isolates obtained from fruits were obtained from five localities in the province of Tungurahua (Ambato, Baños, Patate, Pelileo and Mocha) and one locality in the province of Chimborazo (Riobamba), each isolate presenting different characteristics in both leaf and fruit. The morphological characteristics of isolates of C. fulvum showed an average hyphal size of 93.67 µm in isolates obtained from leaves and an average of 99.18 µm in isolates obtained from fruits. The conidiophores with average length of 27.82 µm in isolates from leaves and average of 25.82 µm in isolates from fruits. In conidiophores with average length of 5.11 µm and average width of 3.67 µm in leaf isolates and average length of 5.8 µm and average width of 4.03 µm in fruit isolates. In the inoculation of healthy leaves and fruits, the infection rate was determined in (%) as the first symptoms appear and as the days go by, the leaves and fruits become more contaminated and obtain a higher infection percentage. In the re-isolation, the same colonies of C. fulvum were obtained in leaves and fruits that were harvested at the beginning of the research.
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    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, Michel
    The 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.
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    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, Michel
    The 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.
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    Aislamiento, caracterización cultural y morfológica de aislados monospóricos del agente causal del moho gris de la hoja de Solanum lycopersicum L. en la provincia de Tungurahua.
    (2022-03) Jácome Pilco, Dayana Estefanía; Leiva Mora, Michel
    The gray mold of the tomato leaf discovered in England in 1883 is characterized by producing yellow areas on the leaves, observing an olive green color on the underside, which affects the agricultural yield and the quality of the fruits. The objective of this work was to obtain monosporic isolates from tomato leaves with signs of gray mold in order to characterize them culturally and morphologically. For the monosporic isolation, the leaf printing method was obtained and later a modification of the Valmas method was obtained. For the cultural characterization of the colonies of the isolates, the following characteristics were evaluated: color of the front and back of the colonies, elevation, texture, edges, pigmentation and presence or absence of perspiration liquid. For the morphological characterization, the microculture method and observation under the microscope of the diameter of the hyphae, the length of the conidiophores and the length and width of the conidia were obtained. For the first time for Ecuador, 80 isolated monospores will be acquired from 8 cantons of the Tungurahua province (Tisaleo, Baños, Patate, Ambato, Cevallos, Píllaro, Mocha and Pelileo) all presenting olive green colors on the obverse of the colony, while in the reverse was black, in addition a superficial elevation, plush texture and irregular shape appeared. Isolates 1, 2, 3, 5, 6, 7, 8, 9, 10 had lobed borders in their colonies, while 5 showed regular borders. no pigment isolated from the culture medium (PDA), nor was there evidence of transpiration liquid in its colonies. The mycelial growth at 7 days was in a range between 0.50 mm.day -1 to 1.29 mm.day -1 , with the average speed being 0.93 mm.day-1 and at 14 days it reached values which were in the range between 0.95mm.day-1 to 1.46mm.day-1 , with the average velocity being 1.23mm.day-1 .The average diameter of the hyphae of the isolates obtained from C. fulvum showed a lower value of 2 µm, a higher value of 11 µm and an average value of 5.41 µm. In the conidiophores, the smallest size was 9 µm while the largest was 116 µm with an average value of 33.01 µm. The conidia regarding the length had a minimum value of 3 µm and the largest value of 13 µm with an average value of 6 µm, while the width of the conidia showed a minimum value of 1.5 µm and the maximum was 5 µm for an average value of 5 µm.