The olive tree (Olea europaea L.), a millenary plant typical of the Mediterranean area, is appreciated for its fruits and oil. It is considered a very rustic species and is generally resistant to abiotic and biotic adversities. However, it can be attacked by various fungal and bacterial pathogens in favorable climatic conditions, which can cause significant production losses. The sanitary defense of the olive grove is an important objective for the economy of the crop as any damage to the plants reduces the vegetative activity and can compromise the fruits, with negative effects on the quality of the finished product and the production costs. Among the fungal pathogens of the olive tree, Spilocaea oleagina (Castagne) Hughes (recently named Venturia oleaginea (Castagne), Rossman et al., 2015) is much feared for the damage it causes in the Mediterranean area (Mekuria et al., 2001; Obanor et al., 2005, Gonzalez-Dominguez et al., 2017, Buonaurio et al., 2023) because this disease, called Peacock’s eye or leaf spot infection, can lead to serious yield loss of approximately 20%. The disease is particularly harsh in densely planted orchards of susceptible olive cultivars and in nurseries (Graniti, 1993; Schena et al., 2011).

The life cycle of the pathogen Spilocaea oleaginea depends on climatic conditions such as temperature and humidity (Obanor et al., 2005; Obanor et al., 2008; Rhimini et al., 2020, Buonaurio et al., 2023). Peacock’s eye is generally associated with a high humidity rate typically occurring during the winter period. During the summer, the disease is not present due to warmer temperatures that prevent germination of the spores of the fungus (Obanor et al., 2011; Rhimini et al., 2020). The growth of the fungus was found to be prevalent in the period from late autumn to spring and less so in the period from the beginning of July to mid-November (Viruega and Trapero, 1999). Peacock’s eye is usually more abundant in the lower parts of the olive trees (Graniti, 1993) and grows on the surface of the leaves; in susceptible cultivars it also severely attacks the fruits (Miller, 1949). The fungus spreads through mobile zoospores. Once in contact with the host cuticle, the conidia germinate and emit a pro-mycelium that perforates it, thus invading the plant tissue into sub-cuticular spaces (sub-cuticular parasites) with the production of a hyphal system. This location is ideal for Spilocaea oleaginea as it derives nourishment necessary for its growth from cell wall degradation, in particular waxes, cutin, lipids, cellulose, and pectin. In addition, the mycelium exploits the presence of the cuticle for protection against dehydration and radiation. The lesions form dark brown round spots (2–15 mm in diameter) that become necrotic and surrounded by concentric yellowish or light brown halos (Graniti, 1993, Buonaurio et al., 2023). These spots, particularly those on the leaf pages, are velvety when the fungus fructifies. On the fruit, when attacked, the fungus manifests itself with brownish spots several millimeters in diameter (Miller, 1949; Lanza et al., 2017). Fruit infection affects ripening, negatively influences oil yield, and causes detrimental injuries to table olives (Salman et al., 2014; Lanza et al., 2017, Buonaurio et al., 2023). Many studies have investigated the role of fungal cutinases in the invasion of plant tissues by enzymatically degrading the cuticle (e. g. Martin and Juniper, 1970; Matsuda et al., 1998).

Currently, only the application of synthetic fungicides (containing copper) allows disease control in the field throughout olive-growing regions of the world (e.g., Teviotdale et al., 1989; Shabi et al., 1994; Obanor et al., 2008; Sistani et al., 2009; Salman et al., 2014). However, the application of chemicals is not desirable in relation to human and environmental health factors. Because the accumulation of pesticides based on copper in the soil causes toxicity in the soil microbiome, in EU countries, the use of copper in olive groves was limited in January 2019 to no more than 28 kg/ha in seven years, at an average of 4 kg/ha/year (EU rule 1981/2018). Therefore, finding a source of genetic resistance represents the only effective way to stop Spilocea oleagina disease (Anton and Laborda, 1989).

Some olive cultivars exhibit low susceptibility to Spilocea oleagina and show an increase in the content of polyphenols and oleuropein derivatives, hydroxytyrosol, rutin, hydroxycinnamic acids, and flavonols having a fungitoxic effect (Mekuria et al., 2001; Báidez et al., 2007; Rahioui et al., 2009; Aabidine et al., 2010); however, the defense mechanisms involved are not well understood. Benitez et al. (2005), screening a resistant cultivar and a susceptible cultivar using the different display method (DD), demonstrated that olive resistance to S. oleagina is genotype-dependent and that an active defense response involving the accumulation of hydrogen peroxide (H₂O₂) leads to the synthesis of Salicylic acid and other hormones showing complex overlaps with wound responsive gene pathway.

A recent histochemical investigation on the possible involvement of a hypersensitive response (HR) conducted by Lanza et al. (2017) demonstrated that olive shows a weak plant defense reaction, limited to the local activation of polyphenol oxidase (PPO)-catalyzed phenolic oxidation in just a few upper epidermal cells, with no HR. Lanza et al. (2017) also speculated that S. oleagina may have evolved a system that enables it to evade the plant defense system due to its subcuticular localization and probably due to a molecular mechanism of simultaneous degradation and uptake of cutin monomers that avoid detection by plant receptors. In this way, the fungus can persist and proliferate within the thin layer of the cuticle without triggering a massive defense response by the host. However, deeper analysis at the molecular level is needed to verify this hypothesis (Lanza et al., 2017).

Nowadays, analysis of the transcriptome using next-generation sequencing (NGS) techniques allows accurate and extensive identification of resistance/tolerance genes and potential biomarkers (Giovino et al., 2015). Scientific research is actively seeking to develop diagnostic methods that make it possible to identify the disease early in the field, before the onset of symptoms (Martinelli et al., 2019), through the analysis of gene expression and the identification of biomarkers in the very early stages of infection. The early-stage identification of the disease could allow prompt intervention to limit the diffusion of the fungal agent; for the reasons given above, it is extremely important to study this further.

This research aimed to discover the possible resistance mechanisms of olive against Spilocea oleagina and to discover genes of resistance and putative biomarkers linked to the infection by analyzing a low susceptible cultivar, Koroneiki (Buonaurio et al., 2023), and a highly susceptible cultivar, ‘Nocellara del Belice,’ using next-generation sequencing approaches.

When a plant is attacked by pathogens it activates a cascade of genes coding for various receptors, effectors, signaling, and defense molecules. The defense response is localized in cells and tissues, and is characterized by rapid cell death at the pathogen ingress site (Balint‐Kurti, 2019). The production and accumulation of pathogen-related (PR) proteins are part of the innate immune response under biotic and abiotic stress (Finkina et al., 2017). These PR proteins accumulate locally in affected areas and surround even non-infected tissues, providing protection. PR proteins are part of the HR or the systemic acquired resistance (SAR) against different types of infections (Liu et al., 2010). PR proteins, described for the first time in Nicotiana tabacum (Van Loon and Van Kammen, 1970) include chitinases, non-specific lipid transfer proteins, β-1,3-glucanases, peroxidases, and thaumatin-like proteins (Van loon and Van strien, 1999; Agrios, 2005; Van Baarlen et al., 2007; Souza et al., 2017; Ali et al., 2018; Vaghela et al., 2022; Wang et al., 2022).

In infected ‘Koroneiki’ leaves, the over-expression of a very high number of PR proteins was evident ( Figures 5 , 6 ; Tables S2 , S3 ), while in ‘Nocellara del Belice’ their expression was more limited.

PR proteins are either extremely acidic or extremely basic and for this reason, they can be highly soluble and reactive (Agrios, 2005). Most PR proteins with an acidic nature are mostly secreted into the extracellular space, whereas PR proteins of a basic nature are mainly located in the vacuole (e.g., Legrand et al., 1987). Some PR proteins have chitinase (Legrand et al., 1987) or β‐1,3‐glucanase activity (e.g., Kauffmann et al., 1987). The enzyme chitinases can hydrolyze chitin, and several are believed to be involved in fungal pathogen defense (Schlumbaum et al., 1986; Kumar et al., 2018). The production of chitinases following fungal infection has often been found in plants, and their function has been validated in transgenic plants in which the expression of cloned chitinase genes has been studied, providing further evidence supporting their involvement in the defense mechanism (Solgi et al., 2015; Kumar et al., 2018; Vaghela et al., 2022). Chitinases hydrolyze chitin/chitosan structural molecules present in various animals and fungi producing small lipo-chito-oligosaccharides (LCOs) (reviewed in Vaghela et al., 2022), which probably function as endogenous stress signal molecules. Interestingly, in infected ‘Koroneiki’ leaves the upregulation of chitinase and β‐1,3‐glucanase genes was manifest. The over-expression of chitinases and β‐1,3‐glucanases in ‘Koroneiki’ T3 therefore suggests the presence of an active antifungal defense system. It can be speculated that the accumulation of chitinases and endoglucanases in infected leaf tissues may interfere with Spilocea oleagina growth, presumably weakening the fungal cell wall and reducing the mycelium progression. Glucans or chitin fragments from the cell wall of Spilocea, peptides/glycoproteins, or fungal Avr may represent the elicitors inducing PR genes.

The infected ‘Koroneiki’ showed the upregulation of many receptor-like kinases, such as Rust resistance kinase Lr10-like, and many LRR receptor-like serine/threonine-protein kinase genes possibly involved in the signaling recognition of pathogen-derived molecules (PAMP). Rust resistance kinase Lr10-like gene in Vitis vinifera is, for example, involved in the recognition of downy mildew (Ricciardi et al., 2022). In addition, a series of signal transduction events, such as lipids and various phytohormones sensing pathogen attack, were also triggered, presumably to activate downstream Spilocea immune defense responses. Generally, plants under pathogen attack produce ethylene, Jasmonate (JA), salicylic acid (SA), and reactive oxygen species (ROS) (Yang et al., 1997). Acidic PRs are upregulated by reactive oxygen species and different types of signaling molecules such as salicylic acid (Epple et al., 1995; Reymond and Farmer, 1998; Vaghela et al., 2022), while PRs of a basic nature (defensin, proteinase inhibitors) are upregulated by methyl jasmonate and gaseous phytohormone ethylene (e.g Xu et al., 1994, reviewed in Vaghela et al., 2022). In ‘Koroneiki’ T3, the upregulation of signaling genes mediated by phytohormones (JA, Brassinosteroids, Auxine, ABA, Gibberellins, Cytokinin, and Ethylene) was significantly induced, as well as six non-specific lipid transfer proteins (nsLTPs), considered antimicrobial peptides (AMPs). nsLTPs genes are involved in defense against pathogens, as demonstrated in many studies on transgenic plants. In transgenic wheat, the over-expression of the lipid transfer protein TaLTP5 increased the resistance to the downy mildew Cochliobolus sativus and the common root rot F. graminearum (Zhu et al., 2012). In transgenic A. thaliana, the over-expression of the lipid transfer protein TdLTP4 gene isolated from T. turgidum increased the resistance against Botrytis cinerea and Alternaria solani (Safi et al., 2015). Furthermore, experiments carried out in transgenic carrot plants showed that it is possible to increase the resistance to Alternaria radicicola and B. cinerea by combining the over-expression of the wheat lipid transfer protein and the barley chitinase 2 gene (Jayaraj and Punja, 2007).

Finkina et al. (2017) proposed a model for the plant defense response mechanism involving nsLTP secretion into the apoplast. Then, nsLTP can bind to other lipid molecules secreted by plants (such as jasmonic acid) or to molecules secreted by pathogens. In this model, when nsLTPs bind to lipid molecules they also interact with LRR serine/threonine protein kinases, as well as with a transmembrane region and a cytoplasmic protein kinase (PK). This interaction causes mitogen-activated protein kinase phosphorylation cascades (MAPK) and possible Ca²⁺-dependent processes, inducing transcription factors and other pathogenesis-related proteins, leading to SAR. This model can fit the molecular scenario observed in ‘Koroneiki’.

Concerning the upregulated TF of cv. Koroneiki (WRKY6-like, WRKY31, WRKY 43, WRKY 53, WRKY70, JUNGBRUNNEN 1-like, MYB family transcription factors (MYB3R-1-like, PHL11, and TF PIF3-like), Ap2-ethylene-responsive transcription factor, and SAR DEFICIENT 1 (SARD1) TF), many have been recognized as key regulators involved in induced systemic resistance (ISR) by simultaneously activating and modulating both the SA and JA/ET signaling pathways (e.g., WRKY70 Jiang et al., 2016; WRKY70 and WRKY53 Hu et al., 2012). Arabidopsis WRKY46, WRKY70, and WRKY53, partially involved in the SA-signaling pathway, positively regulate basal resistance against P. syringae, playing synergetic roles in plant basal defense (Hu et al., 2012; Caarls et al., 2015). However, some WRKY TFs (e.g., WRKY6-like, Benny et al., 2020b) and JUNGBRUNNEN 1-like (Thirumalaikumar et al., 2018) have an important role in combined abiotic and biotic stress responses (Bai et al., 2018). SARD1 is a master transcription factor in plant immunity, induced by SA accumulation. Its over-expression is sufficient to activate downstream defense gene expression (Zhang et al., 2010; Huang et al., 2021).

Extensive crosstalk between the hormone signaling pathways allows transcriptional program fine-tuning, leading to resistance to pathogen attacks (Caarls et al., 2015). In ‘Koroneiki,’ the involvement of abscisic acid and ethylene was evident, which may act synergistically with JA-regulated responses, distinctly suppressing SA responses. Auxin, gibberellins, and cytokinins were also upregulated. The hormonal crosstalk in ‘Koroneiki’ resulted in the regulation of defense signaling pathways and the induction of systemic resistance against Spilocea through the over-expression of many other important R-genes (GDSL esterase lipase, defensin Ec-AMP-D2-like, pathogenesis-related leaf protein 6-like, Thaumatin-like proteins (TLPs) related to the PR5 family, glycine-rich protein (GRP), MADS-box genes, STH-21-like, major allergen Pru ar 1-like, and many proteinases such as metalloendoproteinase 2-MMP-l and aspartic proteinase CDR1-like). GDSL LIPASE1 (GLIP1) is a defense and ethylene-mediated gene that triggers systemic resistance signaling in plants under fungus and microbial attack; it possesses lipase and antimicrobial activities that directly disrupt fungal spore integrity (Oh et al., 2005).

The AMPs called Plant defensins, found to upregulated in ‘Koroneiki’ T3 leaves, have been purified from several plants and their antifungal activity has been demonstrated (Silva et al., 2014; Aumer et al., 2020; Owens and Doyle, 2021; Struyfs et al., 2021). As a part of the host defense system, it seems that they have a multifaceted mechanism of action, depending on the targeted fungal species (Leannec-Rialland et al., 2022), transferring information between innate and adaptive immune systems (Silva et al., 2014). Some defensins necessitate traversing the cell wall and plasma membrane of the fungi to induce their cell death, others exert their toxic effects without entering the fungal cells. (Leannec-Rialland et al., 2022).

Concerning the pathogenesis-related leaf protein 6-like gene, over-expressed in Koroneiki T3, there is much evidence that this class of proteins is an essential defense component against fungi (reviewed in Hoegen et al., 2002)

In ‘Nocellara del Belice,’ the genes DMR6-LIKE OXYGENASE 2-like, DOWNY MILDEW RESISTANCE 6-like (e.g., Zeilmaker et al., 2015), MLO, and alpha carbonic anhydrase (e.g., Zhou et al., 2020) were upregulated and are recognized as susceptibility factors since it has been reported that they may attenuate or even suppress plant immunity in response to changing environmental factors (Zhou et al., 2020). In Arabidopsis thaliana, the inactivation of the DMR6 gene results in increased salicylic acid levels, conferring resistance to diverse pathogens, including bacteria and oomycetes (e.g., Zeilmaker et al., 2015). Nowadays, disruption or inactivation of a single or few susceptible genes is an emergent and faster process to achieve broad-spectrum and durable disease resistance in plants (Acevedo-Garcia et al., 2014; Thomazella et al., 2021). Therefore, in the context of new genome editing technologies (CRISPR/Cas9), these genes may represent possible targets to produce loss-of-function genotypes with improved resistance. TF MYB8, over-expressed in ‘Nocellara del Belice’ infected leaves and downregulated in ‘Koroneiki,’ has been found to be expressed in response to herbivore attacks in other species (Onkokesung et al., 2012).

The Thaumatin-like gene was the unique gene over-expressed in both cultivars. In many species, the over-expression of thaumatin-like protein enhanced resistance to many fungus diseases (Batalia et al., 1996; Benitez et al., 2005; Munis et al., 2010; Acharya et al., 2013; Misra et al., 2016; de Jesús-Pires et al., 2020) as well as to many abiotic stresses (Wang et al., 2022; Su et al., 2021; Sharma et al., 2022). Thaumatin-like proteins possess an acidic cleft that enables them to bind and hydrolyze β-1,3-glucans, conferring antifungal activity (e.g., Wang et al., 2022) or fungal enzyme inhibition (Grenier et al., 1999; Ye et al., 1999).

The RNA-binding protein glycine-rich protein gene, upregulated in ‘Koroneiki’ T3, is known for its anti-fungal and anti-bacterial activity in many species (Czolpinska and Rurek, 2018)

Many proteases, including Metalloendoproteinase genes, were also found to be over-expressed in infected leaves of ‘Koroneiki’; these genes code for proteins involved in the regulation of defense responses against plant pathogen infection (Li et al., 2015). Proteases in other species are reportedly secreted from the cell into the apoplast, a possible site used by pathogens for colonization. According to the latest research by Godson and van der Hoorn, (2021) and Backer et al. (2022), apoplastic proteases can: a) have direct antimicrobial activity; b) activate immune hydrolase; c) be involved in damage-associated molecular pattern release; d) perceive the effectors; e) be involved in the initiation of the HR; and f) be involved in the regulation of both systemic acquired resistance and priming. Furthermore, proteases seem implicated in caspase-like activity in plants, promoting programmed cell death, which is also an important aspect in defense responses (Godson and van der Hoorn, 2021; Backer et al., 2022).

Many genes coding for phenylpropanoid and terpenoid pathways, including those involving lignin, flavonoid, terpene, and phytoalexin production, were found to be over-expressed in ‘Koroneiki’ T3, and to a minor extent in ‘Nocellara del Belice’ T3. Flavonoids, terpenes, and phytoalexins may have antifungal activity and have been shown to play a key role in plant defense against biotic and abiotic stress (Mekuria et al., 2001; Báidez et al., 2007; Rahioui et al., 2009; Aabidine et al., 2010; Giovino et al., 2016; Lanza et al., 2017; Pott et al., 2019; Yadav et al., 2020; Desmedt et al., 2021; Backer et al., 2022). The accumulation of these compounds is more rapid at higher levels in resistant plants, often localized in the infected areas, but is not specific since it may be triggered by many abiotic stresses (Pott et al., 2019; Yousefi et al., 2022).

In addition, many genes encoding for biosynthetic enzymes for cutin, wax, suberin transport, deposition, and remodeling, which are important for epidemical protection against pathogens, were also found to be upregulated in ‘Koroneiki’. Among them, for example, CASPL genes, expressed in the endodermis in a highly tissue-specific manner, encoding for Casparian strip domain proteins (CASPs) induce the formation of the honeycomb-structured lignin brace, which can impede pathogen proliferation (Roppolo et al., 2014; Barbosa et al., 2019).

The Gene Ontology enrichment analysis of all 678 differentially expressed genes in the comparison of ‘Koroneiki’ healthy leaves (T1) with ‘Nocellara del Belice’ T1 revealed that the biological process terms “photosynthesis,” “fatty acid metabolic process,” “lipid catabolic process,” and “response to cold” were significantly enriched ( Figure S2 , Table S3 ). Our study also revealed the enrichment of the GO terms “chloroplast” and “extracellular region,” which demonstrates the plants’ defense response to strengthen the cell wall and increase energy production to support defense-related processes. We also performed GO enrichment analysis on both ‘Koroneiki’ and ‘Nocellara del Belice’ leaves infected by the fungus Spilocaea oleaginea ( Figure S3 , Table S3 ). The analysis showed that there were a very high number of GO terms regulated in ‘Koroneiki’ with respect to ‘Nocellara del Belice,’ which shows its strong resistance against the pathogen.

In our work, an overwhelmingly significant contrast in the number of DEGs between ‘Koroneiki’ and ‘Nocellara del Belice’ infected leaves was evident. There is a weak defense response in cv. Nocellara del Belice, indicated by the lack of signaling genes, limited hormonal crosstalk, limited resistance-related gene expression, and the over-expression of putative susceptibility genes. Only a thaumatin-like gene was over-expressed in both cultivars after infection. The thaumatin-like gene is involved in plant defense against both biotic and abiotic stresses (Wang et al., 2022). By using leaves collected at the beginning of March, we determined that the thaumatin-like gene was also employed to detect the early stage of the disease, and it seems a good candidate for this purpose. However, further analysis is needed to confirm this.

In a similar transcriptomic study conducted to explore the resistance mechanism of two avocado rootstocks under the attack of the fungus Phytophthora cinnamomic, differing in their susceptibility to the pathogen, a tremendously dissimilar genetic response was described, showing the activation of a plethora of genes in the resilient rootstock and the lack of molecular response in the susceptible rootstock (Backer et al., 2022), consistent with our observation in ‘Koroneiki’ vs. ‘Nocellara del Belice’.

Many putative defense genes of cv. Koroneiki, such as chitinase, β‐1,3‐glucanase, nsLTPs, GDSL esterase lipase, defensin Ec-AMP-D2-like, STH-21-like, major allergen Pru ar 1-like,Thaumatin-like, metalloendoproteinase, and Casparian strip domain, as well as some TFs (Ap2-ethylene-responsive, SAR DEFICIENT 1, MYB3R-1-like, PHL11, TF PIF3-like, WRKY70, and WRKY53), can be exploited to develop molecular markers for breeding stress-resistant olive genotypes and can become the target of studies involving genetic transformation and genome editing using CRISPR/Cas9 technologies, aiming to acquire resistance/tolerance to Spilocea oleagina and possibly other biotic/abiotic stresses. The thaumatin-like gene was uniquely over-expressed in both cultivars, and can be used as a possible marker for early detection of the disease. The genes DMR6-LIKE OXYGENASE 2-like, MLO gene, DOWNY MILDEW RESISTANCE 6-like, and alpha carbonic anhydrase were up regulated in cv. Nocellara del Belice and can be further studied to check whether they represent susceptibility factors and to develop biomarkers for screening the germplasm for vulnerability.

The datasets presented in this study can be found in online repositories http://ncbi.nlm.nih.gov/bioproject/PRJNA929711. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

AM, FM, and AG conceived the study; DT, FB, VI, and AM performed the molecular experiments; FB, AM, and DT analyzed the data; BB performed the bioinformatic analyses; AM took the lead in writing the manuscript; TC, AG, FM, and AM. performed funding acquisition. All authors contributed to the article and approved the submitted version.