Discovering the Next-Generation Plant Protection Products: A Proof-of-Concept via the Isolation and Bioactivity Assessment of the Olive Tree Endophyte Bacillus sp. PTA13 Lipopeptides

Abstract

Endophytic microorganisms (EMs) have recently attracted interest for applications in plant protection, mainly due to their bioactive compound-producing capacity. Therefore, we underwent the task of isolating olive tree EMs and investigating their bioactivity against the devastating pathogen Colletotrichum acutatum. Several EMs were isolated; however, the Bacillus sp. PTA13 isolate exhibited the highest toxicity to the phytopathogen. Bacteria of the genus Bacillus exhibit superior bioactive metabolite-producing capacity, with the lipopeptides (LPs) of surfactin, iturin, and fengycin groups being the most studied. A total LP extract and several fractions were obtained, and their bioactivity was assessed against C. acutatum strains. LPs of the major surfactin, iturin, and fengycin groups and the minor gageotetrin and bacilotetrin groups were annotated. The results confirmed the bioactivity of the major LPs, with fengycins being the most fungitoxic. Interestingly, the minor LP fraction exhibited selective toxicity to the fungicide-resistant C. acutatum isolate, an observation that highlights the significance of our approach to comprehensively mine the total LP extract. This work represents a proof of concept of the exploitation of EMs in customized olive tree plant protection and aligns well with strategies that focus on the sustainability and safety of food production via the development of next-generation plant protection products.

Keywords: countercurrent partition chromatography; endophytic microorganisms; fengycins; iturins; natural products; olive anthracnose; plant protection; surfactins.

Figure 1

Confrontation bioassays for the bioactivity assessment of the olive tree bacterial isolate Bacillus sp. PTA13 against Colletotrichum acutatum species complex PLS 90 (wild type) and PLS 88 (resistant to PPPs) isolates. The formation of inhibition zone (red arrows) and the pattern of the fungal culture development were used as indicators of the bioactivity of the endophyte.

Figure 2

LC/ESI/MS total ion chromatograms (TICs) of Bacillus sp. PTA13 lipopeptides (LPs). TIC of (a) the total LP extract, (b) the surfactin-containing upper phase during liquid–liquid extraction, and (c) the corresponding lower phase that contains bacillomycins and fengycins, and the representative TIC of fractions derived from size-exclusion chromatography of the lower phase (see plot c) that contain (d) fengycins and (e) bacillomycins. Annotations of representative LPs are displayed (see Table 1).

Figure 3

MS¹ (upper plots; U) and MS² (middle plots; M) of the annotated Bacillus sp. PTA13 lipopeptides (LP) surfactins C12 and C13, and fengycins A C16 and B C16. The fragmentation patterns were used for the identification of the LP (lower plots; L).

Figure 4

MS¹ (upper plots; U) and MS² (middle plots; M) of the Bacillus sp. PTA13 LP Gageotetrins A–C. The fragmentation patterns were used for the annotation of the LP (lower plots; L).

Figure 5

Effect of the total lipopeptide extract of Bacillus sp. PTA13 on the radial growth of cultures of the olive tree pathogenic Colletotrichum acutatum species complex PLS 90 (wild type) and PLS 88 (resistant to PPPs) isolates. Images of inhibition and observations were taken 17 days post-treatment with N = 3 biological replications. The letters (a–f) above the columns indicate statistical differences between the treatments for each isolate, performing the Tukey HSD test (p > 95%).

Figure 6

Effect of various fractions of the Bacillus sp. PTA13 total lipopeptide extract on the radial growth of the Colletotrichum acutatum species complex isolates PLS 90 (wild type) and PLS 88 (resistant to PPPs). The fractions were applied at concentrations equal to the EC₅₀ of the total LP extract for PLS 90 (27 μg mL⁻¹) and PLS 88 (63 μg mL⁻¹). Observations were taken 10 days post-treatment, and three biological replications were performed per treatment (CPC; Centrifugal Partition Chromatography, L; Lower Phase, S; Biphasic Solvent System, Seph; Sephadex, U; Upper Phase).

Figure 7

Effect of various fractions of the Bacillus sp. PTA13 lipopeptide extract on the radial growth of the Colletotrichum acutatum species complex isolates PLS 90 (wild type) and PLS 88 (resistant to PPPs) displayed using box plots. The fractions were applied at concentrations equal to the EC₅₀ of the total LP extract for PLS 90 (27 μg mL⁻¹) and PLS 88 (63 μg mL⁻¹). Three replications were performed per treatment and the different letters above boxes designate statically significant differences performing the Tukey HSD test (p > 95%) (CPC; Centrifugal Partition Chromatography, L; Lower Phase, S; Biphasic Solvent System, Seph; Sephadex, U; Upper Phase).