Uncovering the antifungal activities of wild apple-associated bacteria against two canker-causing fungi, Cytospora mali and C. parasitica

Abstract

Cytospora canker has become a devastating disease of apple species worldwide, and in severe cases, it may cause dieback of entire trees. The aim of this study was to characterize the diversity of cultivable bacteria from the wild apple microbiota and to determine their antifungal ability against the canker-causing pathogenic fungi Cytospora mali and C. parasitica. Five bacterial strains belonging to the species Bacillus amyloliquefaciens, B. atrophaeus, B. methylotrophicus, B. mojavensis, and Pseudomonas synxantha showed strong antagonistic effects against pathogenic fungi. Therefore, since the abovementioned Bacillus species produce known antifungal compounds, we characterized the antifungal compounds produced by Ps. synxantha. Bacteria grown on nutritional liquid medium were dehydrated, and the active compound from the crude extract was isolated and analysed via a range of chromatographic processes. High-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance analyses revealed a bioactive antifungal compound, phenazine-1-carboxylic acid (PCA). The minimum inhibitory concentration (MIC) demonstrated that PCA inhibited mycelial growth, with a MIC of 10 mg mL^-1. The results suggested that PCA could be used as a potential compound to control C. mali and C. malicola, and it is a potential alternative for postharvest control of canker disease.

Keywords: Pseudomonas synxantha; Antifungal compound; Apple disease; Pathogenic fungi; Phenazine.

Figure 1

A graphical abstract depicting the determination of bacteria-antagonists of pathogenic fungi.

Figure 2

Diversity, abundance, and clustering analysis of cultivable wild apple twig associated bacteria. The Venn diagram indicates the distribution of cultivable bacterial isolates among exo- and endophytes (a). The pie chart depicts the distribution of bacterial isolates. Bacterial species in the yellow and green pie pieces indicate the respective exophytic and endophytic bacterial isolates. The pie chart with a mix of green and orange shows bacterial isolates at the species level that were found both on the surface and inside of twigs (b). In the clustering analysis of wild apple-associated bacteria, the maximum likelihood was inferred using the neighbour-joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (5000 replicates) is shown next to the branches. The evolutionary distances were computed using the Tajima–Nei method and are in units of the number of base substitutions per site. The distance scale represents the number of differences between the sequences. Evolutionary analyses were conducted in MEGA 7 (c).

Figure 3

Relative OTU abundance of wild apple-associated bacteria. The abundance refers to the relative proportion of OTUs containing genera within the distribution of each parent phylum displayed on the x-axis.

Figure 4

Heatmap depicting the cellulase and antifungal activities of microbial isolates obtained from wild apple twigs. Cellulase activity of isolated bacteria (a) and wild apple-associated bacterial species inhibited by nonpathogenic and pathogenic fungal species (b).

Figure 5

Mass spectra and chemical structure of antifungal PCA.

Figure 6

Minimum inhibitory concentration of antifungal PCA against the pathogens C. mali and C. parasitica. MICs of the antifungal compounds 6 days postcultivation on PDA plates at 28 °C (a). The effect of antifungal compounds at serially diluted concentrations on fungal growth at various postincubation times. The different letters indicate significant differences according to one-way ANOVA followed by a Fisher’s PLSD post hoc test (p < 0.05) (b).