*2.1. Effect of Olive Stem Extract on Conidial Viability of Verticillium dahliae* 2.1.1. Experiment I: Effect of Olive Cultivars

The results on the effect of OSE from plants of cvs. Frantoio (tolerant), Arbequina (moderately susceptible), and Picual (highly susceptible) at 5, 10, 15, 20, 25, and 50% concentration on the conidial viability of *V. dahliae* showed that the higher OSE concentration, the higher the RGI. Marked differences between the three olive cultivars were detected from OSE at 10%, whereas that from 'Frantoio' showed higher RGI values than that from 'Arbequina' and 'Picual'; however, in these last two cases, the inhibition did not differ significantly. At the highest concentration evaluated (OSE at 50%), RGI values ranged from 68.9 ± 2.9 to 80.5 ± 1.9% for 'Picual' and 'Frantoio', respectively (Figure 1).

**Figure 1.** Effect of olive stem extract (OSE) from healthy olive plants of cvs. Frantoio (tolerant), Arbequina (moderately susceptible), and Picual (highly susceptible) on relative conidial germination inhibition (RGI) of *Verticillium dahliae* isolate V180. Data are the means of 240 conidia (six replicates) per combination of olive cultivar and OSE concentration. Vertical bars represent the standard error of the means. \*LSD bars represent the critical values for comparison at *P* = 0.05.

In addition, significant differences were observed for EC50 between cultivars (*P* = 0.0239). The OSE from 'Frantoio' showed the lowest EC50 value (EC50 = 16.2 ± 0.88) compared to the OSE from the rest of cultivars, whereas OSE from 'Arbequina' (EC50 = 21.7 ± 1.15) and 'Picual' (EC50 = 20.4 ± 1.08) did not differ significantly from one another (Table 1).

### 2.1.2. Experiment II: Influence of Treatments

The influence of the OSE from olive plants of cv. Picual treated with different treatments on conidial viability showed great differences between treatments (Figure 2). In general, the OSE from plants treated with commercial products showed higher RGI values than those from OSE obtained from plants treated with BCAs. Only the OSE from plants treated by root applications with *A. pullulans* AP08, aluminium lignosulfonate, and copper phosphite showed RGI values higher than 80% when they were tested at the highest concentration.


Picual 20.4 ± 1.08 a

**Table 1.** Effective concentrations of olive stem extract (OSE) from cvs. Frantoio, Arbequina, and Picual to inhibit 50% of conidial germination (EC50; μL mL<sup>−</sup>1) of *Verticillium dahliae* isolate V180.

<sup>a</sup> EC50 of conidial germination was calculated as predicted value of the linear regression of the relative germination inhibition (%) over OSE concentration. Values represent the average of 240 conidia (six replicates) per extract (cultivar) and OSE concentration. Means followed by a common letter do not differ significantly according to Fisher's protected LSD test at *P* = 0.05.

**Figure 2.** Effect of olive stem extract (OSE) from healthy olive plants of cv. Picual treated by foliar (F) or root (R) applications with (**A**) microorganisms (biological control agents) and (**B**) commercial products on the relative conidial germination inhibition (RGI) of *Verticillium dahliae* isolate V180. In each graph, data are the means of 240 conidia (six replicates) per combination of compound and OSE concentration. Vertical bars represent the standard error of the means. \*LSD bars represent the critical values for comparison at *P* = 0.05.

There were significant differences for EC50 between treatments (*P* < 0.0001). In general, OSE from treated plants showed significantly lower EC50 values than that from both negative and positive control (EC50 = 21.5 ± 1.43 and 24.9 ± 0.69, respectively), with the exception of OSE from plants treated by spraying with *A. pullulans* AP08 (EC50 = 24.7 ± 1.61), *Phoma* sp. (EC50 = 17.3 ± 0.55), and salicylic acid (EC50 = 22.1 ± 2.05), which did not differ from the controls (Table 2).

**Table 2.** Effective concentrations of olive stem extract (OSE) from cv. Picual treated with several compounds to inhibit 50% of conidial germination (EC50; μL mL<sup>−</sup>1) of *Verticillium dahliae* isolate V180.


<sup>a</sup> All compounds were applied by foliar or root applications in non-inoculated plants. Control (-): non-treated and non-inoculated plants; Control (+): non-treated and inoculated plants with *V. dahliae* isolate V-180. <sup>b</sup> EC50 of conidial germination was calculated as predicted value of the linear regression of the relative germination inhibition (%) over OSE concentration. Values represent the average of 240 conidia (six replicates) per extract (cultivar) and OSE concentration. Means followed by a common letter do not differ significantly according to Tukey's HSD test (*P* = 0.05).
