Results

AMF species composition and frequency of occurrence

In the rhizosphere of the three olive cultivars, spores of six species belonging to five genera of four families of arbuscular mycorrhizal fungi (AMF) were found. The species found were Acaulospora koskei Błaszk, Diversispora globifera (Koske & C. Walker) C. Walker & A. Schüßler, Gigaspora decipiens I.R. Hall & L.K. Abbott, Gigaspora rosea (T.H. Nicolson & N.C. Schenck), Scutellospora calospora (T.H. Nicolson & Gerd.) C. (Walker & F.E. Sanders), and Glomus ambisporum G.S. Sm. & N.C. Schenck. (Fig. 2). The frequency of occurrence (FO) revealed that four species_, Glomus ambisporum, G. decipiens, G. rosea,_ and Di. globifera, were present in all sampling units (FO = 100%), characterizing them as stable and generalist components of the mycorrhizal community associated with the studied olive cultivars. In contrast, S. calospora showed a more restricted distribution (FO = 25.0%), occurring only in samples from the Arbequina and Picual cultivars. Acaulospora koskei exhibited the lowest frequency (FO = 16.7%), being detected exclusively under the Picual cultivar (Table 2).

Fig. 2: Optical microscopy images of spores of arbuscular mycorrhizal fungi (AMF) extracted from the rhizosphere soil of olive trees (Olea europaea L.) cultivated at the Centro Agropecuário da Palma, Universidade Federal de Pelotas, Rio Grande do Sul, Brazil. All spores were mounted on PVLG and PVLG + Melzer's reagent (1:1) and photographed using optical microscopy. AAcaulospora koskei. Scale bar: 28.52 µm. BDiversispora globifera.Scale bar: 28.52 µm. CGigaspora decipiens. Scale bar: 28.52 µm. DGigaspora rosea. Scale bar: 114.5 µm. E Scutellospora calospora. Scale bar: 28.52 µm. FGlomus ambisporum. Scale bar: 28.52 µm

Species | Arbequina | Coratina | Picual | FO (%)
Glomus ambisporum | 58.2 ± 6.3 | 87.8 ± 22.2 | 77.5 ± 16.9 | 100
Gigaspora decipiens | 39.0 ± 9.4 | 11.8 ± 2.4 | 20.0 ± 9.0 | 100
Gigaspora rosea | 21.5 ± 2.9 | 17.2 ± 9.2 | 41.8 ± 20.5 | 100
Diversispora globifera | 16.5 ± 8.2 | 38.8 ± 6.9 | 30.8 ± 10.1 | 100
Scutellospora calospora | 8.0 ± 9.6 | 0.0 ± 0.0 | 2.2 ± 4.5 | 25.0
Acaulospora koskei | 0.0 ± 0.0 | 0.0 ± 0.0 | 8.4 ± 12.3 | 16.7

Spore abundance among cultivars

In all cultivars, G. ambisporum was the most abundant species (Fig. 3). In Arbequina, G. ambisporum exhibited 198% higher sporulation than S. calospora (p < 0.0001) and 126% higher sporulation than D. globifera (p = 0.005), with a marginal difference of approximately 100% compared with G. rosea (p = 0.050). In addition, G. decipiens showed 158% higher abundance than S. calospora (p = 0.0004), whereas no significant differences were detected among the remaining species. In Coratina, G. ambisporum was 201% more abundant than G. decipiens (p < 0.0001) and 163% more abundant than G. rosea (p < 0.0001). Furthermore, D. globifera exhibited 119% higher abundance than G. decipiens (p = 0.014). In Picual, G. ambisporum showed 354% higher sporulation than S. calospora (p < 0.0001), 136% higher sporulation than G. decipiens (p = 0.002), and 222% higher sporulation than A. koskei (p < 0.0001). Comparisons involving _A. koskei _and S. calospora among cultivars should be interpreted with caution because of their extremely low abundance and the associated high standard errors, resulting in non-significant comparisons (p = 1.0000).

The cultivar identity changed significantly in the abundance of three species (Fig. 3). G. decipiens showed a significantly higher spore abundance in Arbequina than in Coratina, with Arbequina showing approximately 232% higher abundance (p = 0.003). As for D. globifera, its abundance was significantly higher in Coratina compared to Arbequina, with Arbequina showing about 57% lower abundance (p = 0.045). In addition, the species G. rosea showed significantly lower abundance in Coratina than in Picual of approximately 59% (p = 0.035). No significant differences were blocked between cultivars for G. ambisporum (all p > 0.44).

Fig. 3: Stacked bar charts showing the relative abundance (%) of arbuscular mycorrhizal fungi (AMF) species per individual sampling unit, grouped by olive cultivar. The horizontal axis represents individual sampling units numbered 1–4 within each cultivar group (Arbequina, Coratina, and Picual, from left to right). The vertical axis represents relative abundance from 0 to 100%. Each color within the stacked bars corresponds to one of the six identified AMF species: Acaulospora koskei (lightest shade, pink), Diversispora globifera (medium purple), Gigaspora decipiens (dark green), Gigaspora rosea (medium green), Glomus ambisporum (dark purple), and Scutellospora calospora (light lilac). In all cultivars, Glomus ambisporum occupies the largest proportion of the bar

Diversity indices and multivariate analysis

The Shannon index (Fig. 4a) showed a non-significant marginal trend, with Coratina exhibiting lower values than Arbequina (Tukey p = 0.076; H' means: Arbequina 1.351 ± 0.125; Coratina 1.089 ± 0.103; Picual 1.350 ± 0.238). The Simpson index (Fig. 4b) showed that the Coratina cultivar presented significantly lower evenness than Arbequina (Tukey p = 0.006; means: 0.593 ± 0.059 vs. 0.707 ± 0.041) and Picual (p = 0.014; D = 0.696 ± 0.074), while Arbequina and Picual did not differ from each other (p = 0.968).

Fig. 4: Graph containing two box plot graphs A Shannon–Wiener diversity index (H') per sampling unit grouped by cultivar (Arbequina, Coratina, Picual). Each point represents one sampling unit. Coratina shows lower and less variable H' values compared to Arbequina and Picual, though differences were not statistically significant, B Simpson dominance index (D) per sampling unit grouped by cultivar. The vertical axis ranges from approximately 0.5 to 0.8. Coratina showed significantly lower evenness compared to Arbequina and Picual indicated by the letter "b" below Coratina points and letter "a" above Arbequina and Picual points

Multivariate analysis (Fig. 5) revealed that cultivar identity explained 56.2% of the total variation in AMF community composition (PERMANOVA: F2,9 = 5.772; R2 = 0.562; p = 0.004). The homogeneity of the multivariate dispersion was confirmed (betadispersion: F2,9 = 1.774; p = 0.224). NMDS ordination (stress = 0.055) visually corroborated the separation between cultivars, with Coratina forming the most cohesive internal cluster (Fig. 5). Paired PERMANOVA indicated that Arbequina differed significantly from Coratina (F = 12.875; R2 = 0.682; p = 0.033) and from Picual (F = 4.956; R2 = 0.452; p = 0.033), while Coratina and Picual did not differ significantly from each other (F = 2.666; R2 = 0.308; p = 0.154).

Fig. 5: Ordination by non-metric multidimensional scaling (NMDS) based on Bray–Curtis dissimilarity among rhizosphere sampling units of olive trees (n = 12). Points represent individual sampling units; diamonds represent cultivar centroids; ellipses delimit the 95% confidence interval per group. Stress = 0.055

Species contribution to dissimilarity

The SIMPER analysis (Fig. 6) is organized by the main species responsible for the dissimilarity between cultivars. In contrast to Arbequina and Coratina, G. ambisporum (30.4%; p = 0.016) and G. decipiens (cumulative contribution of 59.7%;_ p_ = 0.002) were the main contributors, followed by D. globifera (cumulative 83.9%; p = 0.007). In the Coratina–Picual contrast, G. rosea accounted for 33.7% of the dissimilarity (p = 0.026). No species reached statistical significance in the Arbequina–Picual contrast, a result consistent with the non-significant paired PERMANOVA for this pair.

Fig. 6: Contribution of arbuscular mycorrhizal fungi (AMF) species to Bray–Curtis dissimilarity between olive cultivar pairs, based on SIMPER analysis. Color intensity indicates the percentage contribution of each species to the dissimilarity of the respective cultivar pair. Glomus ambisporum and Gigaspora decipiens were the primary drivers of dissimilarity between Arbequina and Coratina, while Gigaspora rosea was the main contributor to the Coratina and Picual contrast