**1. Introduction**

Verticillium wilt of olive (*Olea europaea* subsp. *europaea*; VWO) causes high levels of tree mortality and reduces fruit yield in most olive-growing areas worldwide and is considered the main limiting factor of olive in Mediterranean-type climate regions [1–3]. In southern Spain, the disease is one of the major concerns for olive growers. Although the global disease incidence in this region is around 0.5%, it can reach values higher than 20%, together with high levels of disease severity and tree mortality in certain areas across the Guadalquivir valley. The causal agent of VWO is the hemibiotrophic soil-borne fungus *Verticillium dahliae*, from which two populations, defoliating (D) and nondefoliating (ND) pathotypes, have been identified in olive, with D pathotype causing the most severe damage [1,4]. The pathogen develops microsclerotia (MS), which are dormant structures that not only confer the ability to survive in the soil for a long time, but also serve as the primary inoculum source in natural infections [1,2,4]. Regarding the life cycle of *V. dahliae*, MS germinate by the stimuli of the root exudates from the susceptible hosts, giving rise to infectious hyphae that penetrate the plant roots and grow until they reach the vascular system. Then, the xylem vessels of the infected plants are colonized by the pathogen

**Citation:** López-Moral, A.; Agustí-Brisach, C.; Leiva-Egea, F.M.;

Trapero, A. Influence of Cultivar and Biocontrol Treatments on the Effect of Olive Stem Extracts on the Viability of *Verticillium dahliae* Conidia. *Plants* **2022**, *11*, 554. https://doi.org/ 10.3390/plants11040554

Academic Editor: Artur Alves

Received: 2 January 2022 Accepted: 18 February 2022 Published: 20 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

through mycelia and conidia development, contributing to the occlusion of the vascular system, as well as the production of gels and tyloses in the xylem vessels. Altogether cause a reduction in water flow, leading to water stress, and, consequently, plants become wilted and eventually die [5–7].

The innate biology of the pathogen, besides of the agronomical factors related to the intensification of olive crop, favours a year-by-year increase in disease incidence and have made it difficult to control VWO, making this disease one of the largest threats to olive groves worldwide [1,8,9]. Likewise, no truly efficient method to control VWO has been reported. Thus, there is no doubt that an integrated disease management (IDM) strategy is needed to prevent *V. dahliae* infections by both pre- and post-planting control measures in olive groves [1,2,10].

Considering IDM against VWO, both genetic resistance and biological control methods must be combined to achieve synergistic effectiveness to reduce both pathogen dispersal and disease incidence in the field. It is well known that disease severity varies depending on the olive cultivars, with the selection of cultivar being essential to avoid serious infections in the field. For instance, 'Picual' is considered one of the most susceptible olive cultivars, whereas 'Arbequina' and 'Hojiblanca' have shown moderate susceptibility, and 'Changlot Real', 'Empeltre', and Frantoio have high levels of tolerance [11,12]. On the other hand, important advances in biological control against VWO have been achieved in the last two decades [2,13]. In this regard, a broad diversity of essential oils, organic amendments, and biological control agents (BCAs), including endophytic bacteria and fungi, plant biostimulants, and host resistance inducers, have been evaluated both under controlled and natural field conditions towards selection of the best candidates for the control of VWO [3,14–18]. Foliar or root applications with two beneficial microorganisms, the fungus *Aureobasidium pullulans* AP08 and the bacterium *Bacillus amyloliquefaciens* PAB-024; and two phosphonate salts, one of copper and the other of potassium, were effective in reducing disease progression in artificially inoculated olive plants [3].

Due to the different behaviour patterns previously observed on the effect of all these treatments against VWO, their mechanisms of action have been explored, elucidating both direct effects, such as dual culture assays and *in vitro* sensitivity tests [3], and indirect effects, such as the effect on root exudates inhibiting MS and conidia germination enhancing the natural plant defence response [7,19] on the viability of the infectious structures of *V. dahliae* and VWO progress. These previous studies contributed to better understanding of how these biocontrol products can act against the pathogen; however, knowledge generated here opens new paths to be explored concerning such a topic. In this way, determining whether these products can modulate the effect of endosphere contents of the treated olive plants on *V. dahliae* infection would suppose other relevant knowledge towards to better elucidating their mode of action.

The xylem vessels are an ideal niche for microbial endophytes such as the Verticillium wilt pathogen by providing an effective internal pathway for whole-plant colonization and by acquiring the scarce nutrients available in xylem sap, either by enzymatic digestion of host cell walls, by invading neighbouring cells, or by inducing leakage of nutrients from surrounding tissues [20,21]. Indeed, xylem sap contains a wide range of compounds beyond water and minerals, such as amino acids [22], organic acids [23], and vitamins [24]. The xylem sap composition of woody plant species, including olive tree, has been characterized in several previous studies [25]. However, this composition can be influenced by multiple factors, such as the water content of the soil [26]; the cultivar; the type and age of organs selected; and the incidence of microbial interactions, including infection by plant pathogens, among others [25]. Related to this aspect, recently, Anguita-Maeso et al. [27] determined that the xylem microbiome of olive plants inoculated with *V. dalhiae* increases the diversity of bacterial communities compared to non-inoculated plants. In addition, these same authors also showed a breakdown of resistance to *V. dahliae* in wild olive trees related to a modification of their xylem microbiome [27]. However, to our knowledge, there are no scientific studies that address the effect of the content of the olive endosphere, including

xylem sap, on the viability of infectious structures of *V. dahliae*. Thus, the aim of this study was to determine the influence of the olive genotype (cultivar resistance) and the interaction between cultivars and biocontrol treatments on the effect of olive stem extract (OSE) on the viability of conidia of *V. dahliae in vitro*.
