Classificació en grups filogenòmics d'aïllats de Pseudomonas fluorescents interessants pel seu ús en protecció de cultiu

Abstract

Traditionally, crop protection for bacterial and fungal diseases has been based on the use of agrochemicals, but some of them have shown harmful effects on the ecosystem and on animal and human health. Moreover, the reduction of active substances that can be use now in plant protection have prompted the development of new alternatives. Biological control is one of the most promising alternatives because it is an environmentally sound and effective means of reducing plant diseases through the use of natural enemies named Biological Control Agents (BCA). Many bacteria pertaining to the Pseudomonas fluorescens group are promising bacteria to be used as BCA, but also as Plant Growth Promoting Bacteria (PGPB). These activities are due to some ecophysiological traits that are distributed phylogenetically among the main groups within this complex. Production of secondary metabolites with antifungal properties, as well as antibiotic or/and plant hormone production is the responsible for these activities. The aim of this work was to carry out a classification into the eight phylogroups described by Garrido-Sanz et al. (2017) of a collection of 51 isolated belonging to P. fluorescens complex used in a previous work by Badosa (2001) in the UdG by means of their new developed PCR based system according to a pattern of presence / absence of nine molecular markers. Firstly, an in-silico test was done to confirm that the primers described in the paper for each molecular marker actually amplified the gene sequence described. Secondly, once the isolates have been recovered from freeze cultures, DNA was extracted using two different protocols. The SIGMA-ALDRICH Kit method showed the best results for obtaining DNA suitable for PCR amplification. Before PCR amplification of the nine molecular markers (DPGf_0, 1, 2, 3, 4, 5, 6, 7 and 8), 16S rDNA amplification was done in order to assure the correct DNA quality for amplification. It also was necessary to optimize the PCR annealing temperatures of the different primer pairs. Finally, according the results obtained with the tested strains we classified 24 P. fluorescens, 8 P. corrugata, 6 P. koreensis, 3 P. protegens, 1 P. chlororaphis, 1 P. jessenii and 1 P. gessardii phylogroups and 7 strains remained without identification. Any strain was classified as P. mandelii. In conclusion, we can state that the method proposed by Garrido et al (2017) is a reliable method to classify strains belonging to the P. fluorescens complex