Aislamiento y caracterización microbiológica, bioquímica y molecular de Agrobacterium Tumefaciens a partir de tejidos vegetales infectados

Abstract

Some pathogen Agrobacterium Tumefaciens wild strains causing crown gall were intended to obtain through the isolation of tumors in roses and its characterization through biochemical and molecular techniques which allow establishing their taxonomy in order to add them to the positive control strain collection at the Laboratory of Molecular Identification (IDgen). The isolation process had two stages and was carried out using roses samples given by IDgen: the first stage had to do with the samples maceration and their direct inoculation into plates with two types of semi-selective media, while for the second stage it was only necessary to use half D1 with a previous stage of selective enrichment. The plates were incubated at 28°C during 3-7 days and their isolates were characterized according to the macroscopic morphology and Gram staining; the strains corresponding to Gram negative bacilli were put under catalase, oxidase, sulfide, motility at 28 and 35°C, acid clearance, potassium tellurium and 3-ketolactose production biochemical tests. Then, it was possible to carry out a chain reaction of polymerase with specific primers for virD2, ipt and ARNr23S regions. From a total of 21 morphologically characterized strains, 17 were Gram negative bacilli and after the biochemical characterization, any of the trains was able to produce 3-ketolactose; 9 were put under molecular characterization; from which, only one (PAT07) reflected an approximate amplicon of 750pb with virD2 and ipt primers, this was amplified and sequenced with universal primers for ARNr 16S, determining an identity of 98.44% for Pseudomonas putida, bacterium which is associated with crown gall in roses, so that it would be appropriate to readjust the isolation criteria as well as the bacterial characterization in order to prevent the development of Pseudomonas spp which interfere the growth of A. tumefaciens.