Antibiose de Gluconacetobacter diazotrophicus contra bactérias diazotróficas e fitopatogênicas de cana-de-açúcar e caracterização molecular de genes envolvidos na síntese de substâncias antagônicas

Abstract

The use of endophytic diazotrophic microorganisms in a sugar cane inoculant may help to decrease the need for nitrogen fertilizer application and in addition may act as biological control agent against pathogens in the crop. Moreover, the antagonism study among these strains may help to elucidate their interactions during the inoculant uses as well as to explore the biotechnological potential. For this, an in vitro antagonism study involving the PAL5T strain was carried out against the bacteria from the sugarcane inoculant developed by Embrapa (Azospirillum amazonense, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans and Burkholderia tropica). In addition, this strain was also tested against Xanthomonas albilineans, causal agent of leaf scald. A double- agar layer method was used with PAL5T strain inoculated at equidistant points of the plates and an overlayer containing liquid culture medium of the bacterium to be inhibited was added. Agents that can induce antagonism, like UV radiation and H2O2 were used as inductors in PAL5T liquid cultures. The induction of bacteriocin synthesis by an external agent such as ultraviolet irradiation or hydrogen peroxide was required to inhibit X. albilineans and H. rubrisubalbicans. The other bacteria were not inhibited by PAL5T grown in the same conditions. In addition, it was studied the role played by the genes possible involved in the production of antimicrobial compounds by the PAL5T strain. Two site-directed mutants defective in synthesis and transport of bacteriocin were constructed with the incorporation of the Tn5 in GDI_0415 and GDI_3499 ORFs, respectively. The ORF GDI_0415 was anoted as a bacteriocin named Linocina M18 while GDI_3499 was anoted as an ABC transporter. The antagonism of the wild-strain and mutants was analyzed by double- agar layer method as previously described. The mutant defective for bacteriocin transport produced the same phenotype as the wild-type strain. The bacteriocin mutant did not show antagonistic activity against the phytopathogen even after UV exposition, suggesting that the bacteriocin (Linocina M18) is responsible for the antagonism against X. albilineans.