Antagonistic effects of Bacillus subtilis subsp. subtilis and B. amyloliquefaciens against Macrophomina phaseolina: SEM study of fungal changes and UV-MALDI-TOF MS analysis of their bioactive compounds

Abstract

The antifungal effect of Bacillus subtilis subsp. subtilis PGPMori7 and Bacillus amyloliquefaciens PGPBacCA1 was evaluated against Macrophomina phaseolina (Tassi) Goid. Cell suspension (CS), cell-free supernatant (CFS) and the lipopeptide fraction (LF) of PGPMori7 and PGPBacCA1 were screened against three different M. phaseolina strains. CS exhibited the highest inhibitory effect (around 50%) when compared to those of CFS and LF, regardless of the fungal strain studied. The synthesis of lipopeptides was studied by UV-MALDI TOF. Chemical analysis of Bacillus metabolite synthesis revealed that surfactin and iturin were mainly produced in liquid medium. Potential fengycin was also co-produced when both Bacillus were cultivated in solid medium. In co-culture assays, the bacterial colony-fungal mycelium interface at the inhibition zone was evaluated by both scanning electron microscopy (SEM) and UV-MALDI TOF, the former to determine the structural changes on M. phaseolina cells and the latter to identify the main bioactive molecules involved in the inhibitory effect. PGPBacCA1 produced surfactin, iturin and fengycin in the inhibition zone while PGPMori7 only produced these metabolites within its colony and not in the narrow inhibition zone. Interestingly, SEM revealed that PGPBacCA1 induced damage in M. phaseolina sclerotia, generating a fungicidal effect as no growth was observed when normal growth conditions were reestablished. In turn, PGPMori7 inhibited the growth of the Macrophomina mycelium without fungal injury, resulting only in a fungistatic activity. From these results, it was determined that the two bacilli significantly inhibited the growth of an important phytopathogenic fungus by at least two different mechanisms: lipopeptide synthesis and competition among microorganisms.

Interaction between bacterial biocontrol-agents and strains of Xanthomonas axonopodis pv. phaseoli effects on biocontrol efficacy of common blight in beans

“…There are also many researches that show good results for bean diseases such as Macrophomina phaseolina (Torres et al, 2016), X. axonopodis pv. phaseoli (Zanatta et al, 2007;Sallan, 2011), Pseudomonas savastanoi pv.…”

Efecto de biocontrol e inhibición germinativa de Bacillus spp. sobre zoosporas de Phytophthora capsici

“…Torres et al (2016) reportaron una inhibición del 56.5±2.1% sobre el crecimiento micelial de Macrophomina phaseolina mediante la técnica de cultivo dual, utilizando suspensión celular de B. amyloliquefaciens. De acuerdo con lo observado en el trabajo de Torres et al (2016) y en el presente trabajo, se demuestra que la capacidad biocontroladora de B. amyloliquefaciens se ve afectada por la naturaleza del patógeno, ya que al confrontar la suspensión celular de B. amyloliquefaciens con zoosporas de P. capsici en medio de cultivo líquido, se observa una inhibición germinativa del 88.15%.…”

“…Por otro lado, Chen et al (2016b), reportaron una tasa de germinación de zoosporas de P. capsici del 100% al ser confrontadas con filtrados obtenidos de un cultivo de Streptomyces plicatus, donde los ingredientes del medio de cultivo fueron papa y sucrosa; sin embargo, los resultados que obtuvieron al utilizar filtrados provenientes de medios de cultivo and B. thuringiensis, respectively. Torres et al (2016) reported 56.5±2.1% inhibition of Macrophomina phaseolina mycelial growth using the dual crop technique and cell suspension of B. amyloliquefaciens. According to Torres et al (2016) and to the present study, it has been proven that B. amyloliquefaciens biocontrol capacity is affected by the pathogen's nature, because when the cell suspension of B. amyloliquefaciens was mixed with P. capsici zoospores in a liquid culture medium, it caused 88.15% germination inhibition.…”

“…(Zhang et al, 2010). El género Bacillus ha sido más estudiado dentro del control biológico y las especies de este géne-ro se consideran candidatos ideales para el control de enfermedades, debido a su potencial antagó-nica (Zhao et al, 2013;Torres et al, 2016). Los mecanismos de control incluyen la producción de has led to the emergence of resistant isolates and environmental pollution (Qi et al, 2012).…”

“…(Zhang et al, 2010). The Bacillus genus has been studied more as part of biological control, and species of this genus are considered ideal candidates for controlling diseases due to their antagonistic potential (Zhao et al, 2013;Torres et al, 2016). Control mechanisms include the production of antibiotic and lithic enzymes, physical and chemical interference, competition, host resistance induction, hyperparasitism and predation (Pal and Gardener, 2006).…”