Antagonistic activity of Bacillus subtilis subsp. subtilis against Setophoma terrestris

ALBARRACIN ORIO A; BRÜCHER, E; SAYAGO, P; DUCASSE, DA

La Falda, Córdoba, Argentina

Workshop; II Latin American PGPR Workshop; 2014

The use of beneficial rhizobacteria offers an attractive way to replace chemical fertilizers, pesticides, other supplements and can also help to manage plant diseases. Onion is the second vegetable crop produced worldwide and Argentina exports onion to over 30 countries including Brazil, United States and the European Union. Many diseases limit production of onion. Pink Root is among the major diseases of onion and its causal agent Setophoma terrestris is one of the most severe pathogens in soils of tropical and subtropical climates. The management of Pink Root is difficult and the strategies currently used are ineffective, there are not plant resistant varieties and the chemical control is not technically or economically feasible mainly because of the risk of degradation products in soil and the high doses used. So, we decided to address the biocontrol as a mean to control the main limiting pathogen of onion production in Argentina. Our objective was to prove the efficiency of a strain of Bacillus subtilis subsp. subtilis, isolated from the rhizosphere of onion, against S. terrestris in vitro. We collected soil samples from fields under continuous onion culture in EEA INTA Hilario Ascasubi, Buenos Aires, Argentina. Bacillus subtilis subsp. subtilis was isolated from the rhizosphere of onion by shaking the soils samples in sterilized water for 20 min and plating the suspensions on LB agar plates. The identification of the bacterial isolate was carried out by MALDI-TOF. The activity of B. subtilis subsp. subtilis against S. terrestris was screened by dual cultures on PDA plates. Antagonism was evaluated by measuring the radial growth of the pathogen after 3-4 days of incubation at 28ºC and the antifungal activity of the cellfree supernatant of B. subtilis culture was measured. Potential morphological alterations in the pathogen grown in dual cultures for 15 days were analyzed by electron microscopy. Bacillus subtilis subsp. subtilis isolated from rhizosphere of onion showed a strong capacity of growth inhibition of Setophoma terrestris in vitro (55% to 64.7%). We found that the cell-free supernatant of the bacterial culture had antagonistic activity against S. terrestris. Interestingly, we observed a high fungal growth inhibition on plates containing cell-free supernatant of B. subtilis previously grown in the presence of the fungus. No significant differences in the fungal growth were obtained between control plates and plates containing cell-free supernatant from B. subtilis grown without previous contact with S. terrestris. Electron microscopy of S. terrestris from co-cultures plates revealed thickened, tortuous or coiled fungal hypha, with deposits and granules and globular like terminations. These results suggested that the strain of B. subtilis under study has a strong biocontrol activity against S. terrestris and that it would be acting diffusible bacterial inhibitory compounds. Moreover, the secretion of antifungal metabolites seemed to be inducible by the presence of the pathogen. The antagonistic compounds caused several alterations in S. terrestris mycelium morphology.