Ciencias Agropecuarias
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Item Aislamiento, caracterización cultural, morfológica, patogénica e identificación de genes AVR en Cladosporium fulvum Cooke.(2023-12) Sánchez Ortiz, Aldo Martin; Leiva Mora, MichelGray 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.Item Caracterización molecular de genes de avirulencia del agente causal del moho gris de la hoja de Solanum lycopersicum L en la provincia de Tungurahua(2022-08) Flores Yanchaliquin, Diana Carolina; León Gordón, Olguer AlfredoCladosporium fulvum or leaf mold is a pathogen described by Cooke in 1883 that develops easily in tomato crops due to favorable temperature and humidity conditions. For the present investigation, the objective was to "Molecularly characterize avirulence genes of the causal agent of the mold of the leaves of Solanumlycopersicum L in the province of Tungurahua". For the molecular characterization of genes, fungal DNA was extracted from each canton (Ambato, Baños, Cevallos, Mocha, Patate, Pelileo, Píllaro, Tisaleo), which consisted of lyophilizing the mycelium, crushing and placing the respective buffers (Buffer A Naoh + Tween 20 and Buffer B Tris Hcl + Edta) and the extracted DNA was stored at -20°C. For amplification using conventional PCR, the amplification conditions for each Avr andEcp and their process were standardized, using dNTPs, Taq DNA Polymerase,forwrad-reverse primers, and ultrapure water with 25 μl of reaction. For visualization,1% agarose gels were made with the running buffer TAE 1X and a 100bp molecular marker (Invitogen) was used. Having as results the presence of the Avr2 and Avr4E avirulence gene, thus also verifying the absence of Avr4 and Avr9, while for extracellular proteins Ecp2, Ecp4 and Ecp5 are presented so that for Ecp1 it does notrecognize the presence of the gene