Diffusible and volatile organic compounds produced by avocado rhizobacteria exhibit antifungal effects against Fusarium kuroshium

Abstract

Rhizobacteria emit bioactive metabolites with antifungal properties that could be used for biocontrol of fungal diseases. In this study, we evaluated the potential of diffusible and volatile organic compounds (VOCs) emitted by avocado rhizobacteria to inhibit the growth of Fusarium kuroshium, one of the causal agents of Fusarium dieback (FD) in avocado. Three bacterial isolates (INECOL-6004, INECOL-6005, and INECOL-6006), belonging to the Bacillus genus, were selected based on their capacity to inhibit several avocado fungal pathogens, and tested in antagonism assays against F. kuroshium. The three bacterial isolates significantly inhibited F. kuroshium mycelial growth by up to 48%. The composition of bacterial diffusible compounds was characterized by the analysis of EtOAc and n-BuOH extracts by using ultra-performance liquid chromatography (UPLC) coupled to high-resolution mass spectrometry (HRMS). The three bacterial isolates produced cyclo-lipopeptides belonging to the iturin, fengycin, and surfactin families. The antifungal activity of n-BuOH extracts was larger than that of EtOAc extracts, probably due to the greater relative abundance of fengycin in the former than in the latter. In addition, isolates INECOL-6004 and INECOL-6006 significantly inhibited F. kuroshium mycelial growth through VOC emission by up to 69.88%. The analysis of their VOC profiles by solid phase micro-extraction (SPME) coupled to gas chromatography and mass spectrometry (GC-MS) revealed the presence of ketones and pyrazine compounds, particularly of 2-nonanone, which was not detected in the VOC profile of isolate INECOL-6005. These results emphasize the need to further investigate the antifungal activity of each bioactive compound for the development of new formulations against fungal phytopathogens.

Keywords: Bacillus; Bacterial lipopeptides; Biological control; Fusarium dieback; Persea americana.

Fig. 1

Maximum-likelihood tree showing the relationship of isolates INECOL-6004, INECOL-6005, and INECOL-6006 with other bacterial strains from the genus Bacillus. Values above nodes are bootstrap values obtained from 1000 replicates

Fig. 2

Antifungal activity of diffusible compounds secreted by bacterial isolates INECOL-6004, INECOL-6005, and INECOL-6006 against Fusarium kuroshium. a Inhibition percentage of mycelial radial growth of Fusarium kuroshium by bacterial diffusible compounds. b Alterations in hyphae induced by bacterial diffusible compounds observed with confocal laser scanning microscopy (× 100). Bars in a represent the mean of three replicates ± s.e. Asterisk symbol represents significant difference in the mycelial growth of the fungus growing with bacteria in comparison with the control (Dunnett’s test, P ≤ 0.05)

Fig. 3

Antifungal activity of volatile organic compounds (VOCs) emitted by bacterial isolates INECOL-6004, INECOL-6005, and INECOL-6006 against Fusarium kuroshium. a Inhibition percentage of mycelial growth of Fusarium kuroshium by bacterial VOCs. b Alterations in hyphae induced by bacterial VOCs were observed with confocal laser scanning microscopy (× 100). Bars in a represent the mean of three replicates ± s.e. Asterisk symbol represents significant difference in the mycelial growth of the fungus exposed to bacterial VOCs in comparison with the control (Dunnett’s test, P ≤ 0.05)

Fig. 4

Confocal laser scanning microscopy images (× 100) of the effect of n-BuOH and EtOAc organic extracts from bacterial isolates INECOL-6004, INECOL-6005, and INECOL-6006, against Fusarium kuroshium. Images of INECOL-6004 were taken at 3 dpi of bacterial organic extracts and images for INECOL-6005 and INECOL-6006 were taken at 6 dpi

Fig. 5

Total counts of iturin, surfactin, and fengycin families determined in n-BuOH and EtOAc bacterial organic extracts from INECOL-6004, INECOL-6005, and INECOL-6006. The number of molecules detected (total counts) was expressed as percentage