Evaluation of the potential agronomic use of different Bacillus strains isolated of wheat (Triticum aestivum) rhizosphere

OLIVELLA, LAURA; BARENGO, PAMELA B.; TROD, B.; MARELLIS, BELKIS E.; FIGUEROA, SOL A.; TORRES MANNO, MARIANO A.; PETITTI, T.; ESPARIZ, MARTIN; DAURELIO, LUCAS D.

Cordoba

Congreso; XVIII Congreso Argentino de Microbiología Genera (SAMIGE); 2022

SAMIGE

The rhizosphere supports a vastly diversified microbial community, including microorganism capable of regulating the development and activity of plants, with potential impact in the yield improvements in crops. Wheat is the main produced wintry crop in our country. Previously, a novel group of Bacillus strains were isolated from wheat rhizosphere some of which, used individually, demonstrated the ability of promote plant growth, including an enhancement of yield. However, in order to be used in formulations, these Bacillus strains should not have cytotoxicity, and in order to be combined, they should be compatible with each other. In this context, the aim of this work was to evaluate the compatibility between B. cereus (ZAV-W77), B. velezensis (ZAV-W70) and B. megaterium (ZAV-W64) strains and their possible cytotoxic effect on eukaryotic cells. In order to analyze their compatibility, growth curves were made with BioTek Synergy ® equipment in plates of 96 wells, where ZAV-W77, ZAV-W70 and ZAV-W64 were combined with the filtrated supernatant (FS) of the other two, FS-77, FS-70 and FS-64 respectively. To obtain the FS, the strains were grown in dextrose potato medium (PD) at 28 °C 48 h, then filtrated with sterile syringe filters of 0.2 μm Minisart®. The assay was made by triplicate and for tree dilutions in Luria Bertani medium (LB)for each filtrated supernatant (1/2; 1/4 and 1/8) and the equipment was set to measure every 20 minutes at 30 °c for 16 hours. The negative control of growth was a mixture of each filtered supernatant with LB, and the positive control was a mixture of a suspension of each strain (108 UFC/ml) and LB. No growth inhibition was observed of strains ZAV-W64 or ZAV-W70 when different dilutions of FS-70 or FS-64 were added, and no inhibition was observed for ZAV-W77 and ZAV-W70 whit different dilutions of FS-70 or FS-77, respectively. In contrast, even when Zav77 was not inhibited bay FS64, strong growth inhibition of B. megaterium ZAV-W64 strain was observed in liquid medium when it was cultured in the presence of FS-77 even at 1/8 dilution, Also, tests were carried out in Potato Dextrose Agar medium where it was observed that both bacteria affect their growth reciprocally. On the other hand, to determine the cytotoxicity an assay was performed using CHO.K1 cells. 8000 cells/well were seeded in complete Minimum Essential Medium (MEM). After incubation, the culture medium was removed from the wells and 1 vol of each of the dilutions of the supernatants (SN) was to be evaluated or the controls (Culture medium or hydrogen peroxide) after 24 hs of incubation, the viability was observed. It was observed that 1/2 dilutions of ZAV-W77 and ZAV-W70 supernatants showed a decrease in viability compared to culture medium dilutions, while this effect was not observed at greater dilutions. Supernatants of the B. megaterium ZAV64 strain did not show decreased viability at any dilution tested. In conclusion, these results suggest that the three strains could be used in future biological formulations, ZAV-W64 and ZAV-W70 individually or combined, while Zav77 was incompatible with Zav64 and should be excluded.