Aislamiento y caracterización de bacterias de la rizosfera de banano (Musa sp.) en República Dominicana y selección de cepas para el desarrollo de biofertilizantes

Resum

Banana is an important item in Dominican Republic economics as a subsistence crop as well as for its exportation. In the latter case, a major part is produced under organic conditions, which production is dedicated to exportation in an 85 %. The demands for a reduction in the use of synthesis inputs in organic farming have led to look for alternatives to the mineral fertilization and the use of pesticides. One of the main proposed and studied strategies is the use of Plant Growth Promoting Rhizobacteria (PGPR). The overall objective of the work was the development of biofertilizers for banana (Musa sp.) in Dominican Republic by using native bacteria. In order to do so, bacteria associated to banana roots (Musa acuminata AAA) cv. Cavendish, coming from organic producing areas in Dominican Republic, were isolated, characterized and assessed in plant. Two hundred and sixty two strains were isolated in two phases, 114 being isolated in the first phase and the rest in the second one. The 144 isolated in the first phase were identified by the sequentiation of the 16S rRNA gene. Sixty six strains were identified at species level, as they showed similarity level greater than or equal to 99.5 % in comparison to any strain type in the sequence of such gene. Twenty nine showed a similarity level below that figure and the identification at species level could not be claimed, whereas the rest belonged to species complexes that cannot be identified by only the aforementioned gene. The 114 isolates belonged to 20 different genera, being Bacillus, Pseudomonas and Enterobacter the majority ones. Different indicator parameters of Plant Growth Promotion (PGP), like production of the hormone indoleacetic acid (IAA), siderophores production, 1-Aminocyclopropane-1- carboxylic acid (ACC) deaminase activity, solubilization of mineral phosphate, atmospheric nitrogen fixation and the control of the fungus Mycosphaerella fijiensis Morelet (black sigatoka) by antagonism, were in vitro assessed in the complete collection. It was observed that, although certain taxonomic groups show trends of greatest expression of some PGP properties – such as IAA production in Pseudomonas or Enterobacter genus and N fixation by free-living Bacillus – in most of the cases they are strain-dependent characteristics. Among the first-phase isolated strains, 5 endophyte and belonging to safe taxa were selected for plant assays under two different production systems: i) production of plant intended for transplanting (plant nursery) and ii) field fruit production. The selection was based on the results of the in vitro PGP trials, by a Principal Component Analysis (PCA), where siderophores production was prioritized. The selected strains belonged to Pseudomonas genus. Two strains of P. plecoglossicida significantly improved the plant growth in the plant nursery and one of them, also increased the fruit production in the field assay. Such strain also produced a significant delay in the xviii infective process of black sigatoka fungus in the field, which was attributed to phenomena of induced systemic resistance. An assay under controlled environment, in growth chamber, with 26 strains selected according to the aforementioned in vitro properties was also carried out, with the objective of evaluating the possible induction of systemic resistance in banana plants against black sigatoka. It was observed that some strains of the Bacillus genus reduced the severity index of the pathogen infection, even more than the P. plecoglossicida strain that produced good results in the field. Another objective of the work was to select the most suitable molecular marker for a preliminary grouping of the isolates, in order to sequence the 16S rRNA gene in the minimum number of isolates. Such grouping is useful when working with large and diverse collections. PCR-RFLP of the 16S rRNA gene and RAPD techniques were tried. The first one gave better results, though it was not possible to group the isolates at the species or subspecies level. Instead, they were grouped at the genus level, and in some cases, some groups consisted of narrowly related genera. For this reason, it was proposed to use the PCR-RFLP technique first, following the RAPD technique within each single PCR-RFLP model. However, sometimes strains of the same species show very different RAPD patterns, which overestimates the real number of species in the collection. This work allows us to conclude that the PGPRs are a valid alternative to mineral fertilizers and pesticides in banana, as they can increase the plant yield, and they reduce the effect of the pathogen black sigatoka. We have also selected specific strains to be used as biofertilizers in banana. Presumably they could be useful even in different Caribbean places, because there are not important variation between the bacterial populations among the sampling localities.