2.1. Verticillium dahliae–olive bioassays

The V. dahliae isolate V937I [34], representative of the highly-virulent, D pathotype (linage 1A) [35] was used. The V937I inoculum (conidia suspension) was prepared as described by Jiménez-Ruiz and co-workers [24]. All tissue samples originated from bioassays performed and described in our previous works [24,25]. These experiments were carried out to assess, at the whole-transcriptome level, the interaction under non-gnotobiotic conditions between olive cultivars differing in susceptibility to VWO and the D pathotype of V. dahliae. Therefore, for this present study, the same infected tissue samples generated by Leyva-Pérez and co-workers [25] were analysed. Briefly, olive plants (cvs. Frantoio and Picual; four-month-old) obtained from a commercial nursery located at Córdoba province (southern Spain) were inoculated by immersing their roots systems in a conidial suspension (1 × 10⁷ conidia mL⁻¹ for 30 min) of the isolate V937I as previously described [36]. ‘Frantoio’ is tolerant to VWO, while ‘Picual’ is very susceptible to D isolates of V. dahliae [9,12,25]. Inoculated plants were then individually transplanted into polypropylene pots filled-in with an ad hoc prepared autoclaved sandy substrate [37]. Culturing conditions were: random distribution of pots in a growth chamber adjusted to 24 °C (day)/21 °C (night), 60% relative humidity, 14-h photoperiod of fluorescent light (360 µE m⁻² s⁻¹) during 15 days (for RNA sampling) or two months (for VWO symptoms observation). The photoperiod was gradually increased to alleviate plant stress after manipulation, inoculation and transplanting [12,24]. Roots of each plant were harvested at 0, 2, 7 and 15 days (three plants/time point) after V937I inoculation (DAI). Tissue samples were immediately frozen in liquid nitrogen and kept at −80 °C until total RNA extraction. A group of ‘Picual’ and ‘Frantoio’ V. dahliae-inoculated and non-inoculated plants were kept to verify actual VWO symptoms development.