Endophytic Diversity in Sicilian Olive Trees: Identifying Optimal Conditions for a Functional Microbial Collection

Abstract

This study aims to identify the optimal conditions-host, plant material, seasonality, and agricultural practices-for isolating and developing a collection of culturable endophytic microorganisms to support sustainable Olea europaea L. cultivation. Samples were collected from three Sicilian olive cultivars ('Nocellara del Belice', 'Nocellara Etnea', and 'Nocellara Messinese') and six wild olive accessions across different phenological phases and under organic and conventional agronomic management. Endophytes were isolated from leaves and twigs using a culture-dependent approach, and their taxonomic diversity and plant-growth-promoting (PGP) traits were analyzed. A total of 133 endophytic isolates were identified, spanning bacterial (Proteobacteria, Firmicutes, and Actinobacteria) and fungal (Ascomycota and Basidiomycota) phyla. Wild olive trees contributed more than cultivated varieties to enriching the diversity and composition of culturable endophyte collection as well as twigs instead of leaves. Winter sampling allowed to implement the taxonomic genera of olive endophyte collection. Both farming systems favored an increase in the composition of microbial collection, though organic farming systems supported greater microbial richness. Functional analysis highlighted key PGP traits in a selection of bacterial isolates, including indole-3-acetic acid and siderophore production, nitrogen fixation, and antifungal activity. Bacillus spp. dominated enzymatic activities, such as amylase, protease, and lipase production, as well as antifungal activity against the olive fungal pathogen Neofusicoccum vitifusiforme. This research highlights the significant diversity and functional potential of Mediterranean olive endophytes. Our findings emphasize the role of native microbial communities as bio-inoculants, promoting plant growth, nutrient uptake, and disease resistance. These insights lay the groundwork for developing targeted olive-microbial consortia for biocontrol and stress tolerance applications.

Keywords: Olea europaea L. subsp. europaea var. europaea; Olea europaea L. subsp. europaea var. sylvestris; antifungal activity; cv. ‘Nocellara Etnea’; cv. ‘Nocellara Messinese’; cv. ‘Nocellara del Belice’; olive microbiota; plant growth-promoting microorganisms.

Figure 1

Box plots showing total microbial counts (log₁₀ CFU/g) of leaf (A,C) and twig (B,D) endophytes isolated from the different cvs. ‘Nocellara del Belice’ (NB), ‘Nocellara Etnea’ (NE), and ‘Nocellara Messinese’ (NM) and Sicilian wild olive trees (SYLV) during the four phenological phases (WD, winter rest; FL, flowering; FS, fruit set; MAT, fruit maturation) (A,B) and farmed under organic (ORG) and conventional (CONV) management and no agricultural practices (NONE) (C,D). The central horizontal bars are the medians. The central symbol X corresponds to average values, and the red vertical lines are the standard deviations. Points above and below the whiskers’ upper and lower bounds are outliers.

Figure 2

Relative abundance of endophytic bacterial genera in hosts (A), phenological stages (B), plant organs (C), and farming systems (D) of olive trees cvs. ‘Nocellara del Belice’ (NB), ‘Nocellara Etnea’ (NE), and ‘Nocellara Messinese’ (NM) and Sicilian wild olive trees (SYLV).

Figure 3

Relative abundance of endophytic fungal genera in hosts (A), phenological stages (B), plant organs (C), and farming systems (D) of olive trees cvs. ‘Nocellara del Belice’ (NB), ‘Nocellara Etnea’ (NE), and ‘Nocellara Messinese’ (NM) and Sicilian wild olive trees (SYLV).

Figure 4

Venn diagram of shared and specific microbial genera from different cvs. ‘Nocellara del Belice’ (NB), ‘Nocellara Etnea’ (NE), and ‘Nocellara Messinese’ (NM) and Sicilian wild olive trees (SYLV); (A) phenological phases (B), plant organs (C), and farming systems (CONV, conventional; ORG, organic) (D).

Figure 5

Representation of the dual culture assay showing antifungal activity of Bacillus licheniformis Bl_SYLV02R and Trichoderma harzianum S3 against the fungal pathogen Neofusicoccum vitifusiforme (Nv-P3) and how the inhibition was measured. R_c represents the longest distance of fungal mycelium growth from the inoculated fungal plug, and R_exp is the horizontal distance from the inoculated fungal plug towards the screened isolates.