Novel Pseudomonas Species Prevent the Growth of the Phytopathogenic Fungus Aspergillus flavus

Abstract

In response to the escalating demand for sustainable agricultural methodologies, the utilization of microbial volatile organic compounds (VOCs) as antagonists against phytopathogens has emerged as a viable eco-friendly alternative. Microbial volatiles exhibit rapid diffusion rates, facilitating prompt chemical interactions. Moreover, microorganisms possess the capacity to emit volatiles constitutively, as well as in response to biological interactions and environmental stimuli. In addition to volatile compounds, these bacteria demonstrate the ability to produce soluble metabolites with antifungal properties, such as APE Vf, pyoverdin, and fragin. In this study, we identified two Pseudomonas strains (BJa3 and MCal1) capable of inhibiting the in vitro mycelial growth of the phytopathogenic fungus Aspergillus flavus, which serves as the causal agent of diseases in sugarcane and maize. Utilizing GC/MS analysis, we detected 47 distinct VOCs which were produced by these bacterial strains. Notably, certain volatile compounds, including 1-heptoxydecane and tridecan-2-one, emerged as primary candidates for inhibiting fungal growth. These compounds belong to essential chemical classes previously documented for their antifungal activity, while others represent novel molecules. Furthermore, examination via confocal microscopy unveiled significant morphological alterations, particularly in the cell wall, of mycelia exposed to VOCs emitted by both Pseudomonas species. These findings underscore the potential of the identified BJa3 and MCal1 Pseudomonas strains as promising agents for fungal biocontrol in agricultural crops.

Keywords: Pseudomonas; bacteria volatile compounds; biological control; microbial antagonistic activity; phytopathogenic fungus.

Figure 2

Taxonomic affiliation of Pseudomonas sp. strains BJa3 and MCal1. Tree of BJa3 (A) and MCal1 (B) inferred with FastME [63] from Genome BLAST Distance Phylogeny method (GBDP) distances calculated from genome sequences. Branch lengths are scaled in terms of GBDP distance formula d5; numbers above branches are GBDP pseudo-bootstrap support values from 100 replications. The trees were constructed using the TYGS tool.

Figure 1

Effect of bacterial VOCs on A. flavus growth inhibition after 2 days of co-culture. Inhibitory effect of bacterial VOCs from monocultures and mixtures of BJa3 with MCal1. Controls correspond to A. flavus without the bacterial inoculum, E. coli DH5α and Pseudomonas KT 2440. Different letters indicate significant differences between treatments. Standard errors are indicated by vertical lines (Tukey’s test, p-value < 0.05).

Figure 3

Effect of bacterial-diffusible compounds on A. flavus growth inhibition after 2 days of co-culture. (A) Controls (CTRL-) corresponding to A. flavus without the bacterial inoculum, using Vogel, LB, and Vogel + LB medium, respectively. Effects of diffusible compounds in the absence of fungi with the bacteria E. coli DH5α and Pseudomonas BJa3 and MCal1, respectively, on the mycelial growth of the fungus A. flavus are shown in the second line. Effects of diffusible compounds on the mycelial growth of the fungus A. flavus from the bacteria E. coli DH5α and Pseudomonas BJa3 and MCal1, respectively, when cultivated in the presence of the fungus in liquid medium are represented in the third line. (B) Inhibitory effect of soluble bacterial compounds from the 2 selected isolates on the growth of A. flavus mycelium. The asterisk (*) indicates the condition of co-culture of bacteria and fungus in the inoculum. Different letters indicate significant statistical differences between treatments. Standard errors are indicated by vertical lines (Tukey’s test, p-value < 0.05).

Figure 4

Representative confocal laser scanning microscopy of the mycelium of A. flavus. (A) Conidial head of A. flavus, with phialides highlighted by the arrow head. (B) Vesicle and conidia structures are intact and developed, as highlighted by the arrow heads. (C) Developed hyphae with demarcated septa, shown by the arrow heads. Images (A–C) are fungal structures preserved in the absence of bacteria. (D) Conidial head with developed conidia, as highlighted by the white square. (D,F) Preserved and intact hyphae, with septa and intact hyphae structure, as indicated by the arrowheads. Images (D–F) are fungal structures preserved in the presence of volatiles from E. coli DH5α. (G) Fungus exhibits signs of late germination, such as thinner and less developed hyphae, delimited by white rectangle. (H,I) Hyphae with a scaly appearance, as highlighted by the arrowhead and without septa. Images (G–I) are fungal structures damaged in the presence of volatiles from strain BJa3. (J) Late germination of hyphae. (K) Hyphae with internal cell darkening, as highlighted by the arrow head. (L) Hyphae with internal disorganization and absence of septa, as demarcated by the white rectangle. Images (J–L) are fungal structures damaged in the presence of volatiles from strain MCal1.

Figure 5

Analysis of the volatilomes produced by the bacteria BJa3, MCal1, E. coli DH5α and fungus A. flavus. (A) Venn diagram considering only the 47 VOCs that have statistically significant differences in spectra peak area in each group. (B) PCA of the compounds annotated in the volatilome of the bacteria and fungus.

Figure 6

Heatmap of the compounds annotated in the volatilome of the bacteria and fungus. Columns represent each sample condition. Rows represent the different VOCs evaluated. The color code indicates the abundance of each compound (scale from black, low abundance, to yellow, high abundance). The white rectangle indicates the set of volatile compounds produced by the BJa3 and MCal1 bacteria of interest and through the interaction between the bacteria and the A. flavus fungus. The red rectangles show some volatile compounds whose antifungal action has already been described in the literature. The columns were clustered based on Euclidean distance from the peak area average of each sample.