1 Introduction

The genus Verticillium Ness (phylum Ascomycota) includes feared crop pathogens that are common in temperate and sub-tropical regions of the world (Pegg and Brady 2002). Above all, the asexual species V. dahliae Kelb. is considered a major threat in agriculture because it attacks hundreds of herbaceous and tree species of the eudicot clade (Pegg and Brady 2002). This soil-borne pathogen often causes wilting symptoms, a pathological condition characterized by a dysfunction of the xylem in transporting water and hence, a reduced nutrient flow to stem and leaves (Agrios 2005). In woody plants, symptoms often include a sectorial or general decline, stunting, dieback, a relatively rapid insurgence of wilting and (early) discoloration of leaves, as well as browning of the xylem (Agrios 2005). Yield penalties can easily reach 50% for species like cotton, olive, and ornamentals (EFSA Panel on Plant Health 2014). In case of severe infection, verticillium wilt can lead to blockage of the vascular tissue by vessel occlusion, and death of the plant (Pegg and Brady 2002).

Differently from many other plant fungal pathogens characterized by a narrow species-specificity, V. dahliae populations are cross-pathogenic, even if a significant host adaptability is common (i.e., strains are more virulent on host species from which they are isolated) (Bhat and Subbarao 1999; Klosterman et al. 2009). The ability of this vascular pathogen to evolve races able to overcome host resistance can give rise to genotypic-specific races. These are usually distinguished according to the genetic loci that can control the pathogen, as demonstrated for instance in tomato and lettuce (Fradin et al. 2009). Host adaptation can generate differences in virulence (Vallad et al. 2006). A typical example in cotton and in olive is represented by the defoliating (D) and non-defoliating (ND) pathotypes (Hu et al. 2015; López-Escudero and Mercado-Blanco 2011). While the former, characterized by early abscission of asymptomatic leaves, is considered more virulent (Jimenez-Diaz et al. 2012), the latter is more common and diffuse (López-Escudero and Mercado-Blanco 2011).

The ability of the V. dahliae to survive for long periods of time in the soil as microsclerotia, its polyphagia, and the limited efficacy of chemical control measures in curing infected plants, make this pathogen a problem for perennial tree crops like olive. In this plant species concerns are also due to the sanitary conditions of the infected propagation material and to the diffusion of new management approaches, such as irrigation and high-density planting (Jimenez-Diaz et al. 2012). Since its identification on dahlia in the second decade of the last century, V. dahliae was described for the first time on olive in 1946 in Sicily (Pegg and Brady 2002). Currently, the pathogen has been found in virtually every olive growing country, from California to Australia (Schena et al. 2011; EFSA Panel on Plant Health 2014; Pegg and Brady 2002).

With a total production of around 331 thousand metric tons, Italy is the third largest olive oil producing country in the world (after Spain and Tunisia) (https://www.fao.org/faostat; accessed the 1st February 2023). Interestingly, a large part of the Italian olive growing is still based on traditional systems such as rainfed long-lived groves made of large (often monumental) trees planted at low-to-medium densities, frequently in irregular layouts(Caracuta, 2020). Moreover, the Italian production is based on a wealth of local varieties selected by farmers in hundreds of years (Mazzalupo 2012). Their relevance is testified by the various PDO oils and the production of numerous monovarietal olive oils. Each Italian region has its own germplasm, which is often considered irreplaceable and indispensable to guarantee excellence of the olive oil sector, sustainability of rural economies, and conservation of farmed landscape in marginal areas.

The features of the V. dahliae along with the limited efficacy of chemical control strongly favor the implementation of an Integrated Pest Management approach, which also includes the use of resistant cultivars for rootstock selection or the establishment of new orchards as a cost-efficient and virtually environmentally risk-free strategy to prevent the diffusion of pathogens (Bubici and Cirulli 2012; Elliott et al. 1995). This is particularly relevant for the oliviculture in Southern Italy, where grafting (often on seed-propagated rootstocks) is common. Screening for tolerance to V. dahliae has been carried out in comparing wild and cultivated germplasm collected in different countries (López-Escudero et al. 2004; Erten and Yıldız 2011; Jiménez-Fernández et al. 2016; Serrano et al. 2021; Godena et al. 2022). These works indicated that there is sufficient variation in the susceptibility of the O. europaea to verticillium wilt.

The aim of this work was to evaluate the influence of V. dahliae in a group of important varieties of the Campania region of Italy. Specifically, we aimed at assessing the vegetative growth of young olive plants infected with the pathogen considering the shoot extension, the stem diameter growth and the number of leaves. Knowledge of the differences among different varieties during disease progression can be helpful to better understand the resistance mechanisms in olive.

2 Materials and methods

2.1 Plant materials

The experiment was carried out in 2007 on one-year old own-rooted olive (Olea europaea L.) plants of nine cultivars: ‘Biancolilla’, ‘Caiazzana’, ‘Frantoio’, ‘Ogliarola’, ‘Ortolana’, ‘Racioppella’, Rotondella’, ‘Salella’, and ‘Tonda’. While ‘Frantoio’ was chosen as one of the most common and representative Italian olive cultivars, the other eight are important local varieties diffused especially in Southern Italy. As previous works raised concerns about the true identity of the varieties under investigation (e.g., the Spanish ‘Frantoio’-‘Oblonga’ identity) (Barranco et al. 2000; Martos-Moreno et al. 2006), the genetic identity of the mother plants was previously investigated (Rao et al. 2009; Corrado et al. 2011).

2.2 Fungal isolate and inoculation

The non-defoliating strain VdCQ205 of V. dahliae, stored at the Department of Agricultural Sciences, University of Naples Federico II, was used to perform the experiments. VdCQ205 was isolated by olive in Campania region from the cv. ‘Leccino’ and stored at -80 °C in 30% glycerol. The strain was cultivated on Potato Dextrose Agar (PDA; Sigma-Aldrich, Milan, Italy) and transplanted on VIM medium (Hoyos et al. 1991) before inoculation. VIM is a minimal medium that allows to recover a conspicuous amount of microsclerotia, which represent the main natural source of inoculum for the host studied. V. dahliae was grown for 24 days on VIM medium at 23 ± 2 °C in the dark, then the agar medium was pureed in a blender mixer with 200 mL of distilled water until obtaining a homogeneous suspension. The amount of microsclerotia was evaluated using a Thoma’s blood cell counter, adjusting the final concentration at 2 × 103 CFU/mL of suspension. For each cultivar tested, twenty plants per cultivar were extracted from the pots, scrolled to remove the substrate, and then plant roots were carefully abraded to produce light wounds. Thereafter, the roots were dipped in the microsclerotia suspension (described above) for 15 min and transplanted in 20 cm plastic pots filled with a 1:1 v/v mixture of peat and sand, sterilized twice at 100 °C for one hour. Plants inoculated with sterile distilled water represented the non-inoculated control. Pots were finally placed on a plastic mulch sheet covering the experimental area (to be isolated from the soil) and on a large plastic saucer to avoid cross-contamination with leaking water, and completely randomized. Irrigation was carried out regularly, avoiding waterlogging.

At the end of the experiment, tissue from inoculated and control plants were cultured to confirm infection. Stems were cut, washed in tap water, disinfected for twenty minutes in a 1.0% solution of sodium hypochlorite, and rinsed three times in sterile distilled water. After drying under sterile hood, the bark was removed with a scalpel and the tissue fragments were incubated on PDA at 23 ± 2 °C for 7–14 days in the dark (until colonies appeared on the last inoculated tissue sample).

2.3 PCR analysis

The mycelium was manually harvested from cultured PDA plates, lyophilized, and stored at -80°C until analysis. For DNA isolation, approximately 1 g (fresh weight) of lyophilized mycelium was ground to fine powder in liquid nitrogen and then added with 10 ml of DNA Extraction Buffer (0.5 M NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1% SDS). The mixture was thoroughly resuspended, added with 5 mL of phenol and 5 mL of a 25:24:1 phenol:chloroform:isoamyl alcohol solution (PCIA solution). After mixing, samples were spun for 10’ at 4800 rpm at 4 °C in a 5810R centrifuge (Eppendorf, Milan, Italy). The recovered upper phase was then added to the 5 mL of PCIA and centrifuged as above. The clean aqueous phase was treated with 50 µl of a 10 mg/mL RNAse A (Sigma, Milan, Italy) at 37 °C for 30 min. Nucleic acids were precipitated adding 0.6 volumes of ice-cold isopropanol and incubating the samples at 4 °C for 15 min. DNA was recovered by cold centrifugation for 15 min at 4800 rpm. The pellet was washed twice with a 70% ethanol solution, air dried, and then solubilized in 300 µl of 1 x TE solution.

Total genomic DNA was isolated from roots. Roots were cut from the stem, washed with tap water, bleached for two minutes, rinsed with distilled water, cut in pieces (discarding very tough parts), snap-freezed in liquid nitrogen, and shredded in a blender with clean razor blades. The resulting powder was stored at -80 °C until use. DNA isolation was carried out using the DNeasy Plant Mini Kit (Qiagen, Milan, Italy) as per manufacturer’s instructions.

PCR was performed using the primers FVD (5’ GG TCC ATC AGT CTC TCT G) and RVD (5’-TCC GAT GCG AGC TGT AAC) (Karajeh 2006) annealing to the Internal Transcribed Spacer (ITS) 1 sequence of the V. dahliae genome and yielding a 330 bp product. PCR reactions were performed in a 25-µL volume containing 200 ng DNA (for the analysis of the plants) or 10 ng DNA (for the analysis of the pathogen), 1.5 mM MgCl2, 100 µM deoxyribonucleotides, 0.3 µM of each primer, and 1 U Taq DNA polymerase (Promega) in 1× PCR buffer. The thermal profile included a first denaturation stage of 4 min at 94 °C, 35 cycles each made of three steps (94 ° C for 30’’; 56 °C for 30’’; 72 °C for 30 °C), followed by a final extension stage at 72 °C for 9 min. Reactions were performed in a Mastercycler Gradient thermocycler (Eppendorf). Amplification products were separated by agarose gel-electrophoresis in 1 X TAE buffer stained with ethidium bromide (Sambrook et al. 1989) and amplicon size was estimated using as reference the 1 kb Plus DNA ladder (Invitrogen, Milan, Italy).

2.4 Vegetative growth and susceptibility to V. dahliae

The stem diameter, total shoot length per plant, and total number of leaves per plant were measured on three dates: on the day of fungal inoculation, and 53 and 95 later (hereafter 0, 53, 95 days after inoculation, DAI). Stem diameter was measured 5 cm above the substrate surface with a digital caliper, whereas total shoot length per plant was calculated measuring the length of all shoots with a measuring tape.

To measure the difference between the cultivars in the vegetative growth vigor, the relative growth rates of the total shoot length (RGRSL), the total number of leaves (RGRNL), and of the stem diameter (RGRStD) were measured in the control plants as follows.

$$ {RGR}_{SL}=\frac{{SL}_{DAI95} - {SL}_{DAI0}}{{SL}_{DAI0} \times95}$$
(1)

Where SLDAI0 and SLDAI95 are the total shoot length per plant measured, respectively, on DAI 0 and 95, and 95 are the number of days between the two measuring dates.

$$ {RGR}_{NL}=\frac{{NL}_{DAI95} - {NL}_{DAI0}}{{NL}_{DAI0 }\times95}$$
(2)

Where NLDAI0 and NLDAI95 are the total number of leaves per plant measured on DAI 0 and 95, respectively.

$$ {RGR}_{StD}=\frac{{StD}_{DAI95 }- {StD}_{DAI0}}{{StD}_{DAI0} \times95}$$
(3)

Where StDDAI0 and StDDAI95 are the stem diameter measured on DAI 0 and 95, respectively.

In addition to estimate the susceptibility of the different cultivars to V. dahliae, the following two indexes of vegetative growth inhibition (%ISL and %INL) were calculated.

$$ {\%I}_{SL}=\frac{{SL95}_{control }- {SL95}_{inoculated}}{{SL95}_{control}}$$
(4)

Where SL95control and SL95inoculated are the total shoot length per plant measured on DAI 95 in control and inoculated plants, respectively.

$$ {\%I}_{NL}=\frac{{NL95}_{control }- {NL95}_{inoculated}}{{NL95}_{control}}$$
(5)

Where NL95control and NL95inoculated are the total number of leaves per plant measured on DAI 95 in control and inoculated plants, respectively.

2.5 Statistical analysis

The significance of the effect of Time (T), Cultivar (CV), Inoculation (I), and the CV × T, T × I, CV × I, and CV × T × I interaction were assessed using three-way repeated measures ANOVA. The Tukey’s honestly significant difference (HSD) test was used as a post hoc test for mean separation (p ≤ 0.05). Furthermore, differences between cultivars in the vegetative growth vigor and in the susceptibility to the V. dalhliae were studied with a Principal Component Analysis (PCA) using, as input, five variables: RGRSL, RGRNL, RGRStD, %ISL, and %INL. Statistical analyses and graphical representation were done using R 4.2.

3 Results

3.1 Vegetative growth

Following the inoculation, infected plants showed symptoms of disease (such as progressive leaf chlorosis and curling) starting at about three weeks. To quantify the intensity of the fungal inhibition and to follow disease progression, we monitored quantitatively traits such as the growth of the stems (as length and girth) and the number of leaves. There was a significant three-way interaction among the three experimental fixed factors (the cultivar, C; the time following infection, T; the inoculation with the Verticillium, I) over the length of the stem but not for the girth and number of leaves (Table 1). This can be clarified considering that a main inhibitory effect of the Inoculation was not significant for the girth of the stem, and therefore, this parameter it is of little help for monitoring disease progression and resistance. On the other hand, the Inoculation with the pathogen had a significant main effect on the number of leaves but effect on this variable of the categorical factor C did not statistically depend on the Time.

Table 1 Effect of the time, cultivar, inoculation, and their interaction on vegetative traits of on-year old olive cuttings assessed by three-way repeated measures ANOVA. Separately for each source of variation and within each column, means followed by a common letter are not significantly different according to the Tukey test (p ≤ 0.05). Asterisks indicate the level of statistical significance, with three asterisks corresponding to p-values less than 0.001
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Specifically, total plant shoot length was significantly affected by the Time (T), Cultivar (CV), Inoculation (I), and the CV × T, T × I, and CV × T × I interactions (Table 1). Independently of the cultivar and the inoculation, this variable increased on average three-fold between the beginning and the end of the experiment (95 days after inoculation, DAI), which indicated that plants grew following transplant. As expected, there were significant difference among cultivars. Marginal mean of total plant shoot length was highest in ‘Rotondella’ and lowest in ‘Racioppella’ plants (30.1 and 10.7 cm/plant, respectively).

The categorical factor Inoculation inhibited both the total length of the shoots and the number of leaves. Specifically, inoculation induced on average a 39% decrease in total shoot length per plant (Table 1), but the effect of the inoculation with V. dahliae significantly varied depending on the cultivar (Table 1; Fig. 1). Ninety-five days after inoculation total shoot length of the inoculated plants was significantly lower than control in ‘Biancolilla’, ‘Caiazzana’, ‘Frantoio’, ‘Rotondella’, and ‘Tonda’, but the intensity of this effect in shoot extension growth depended on the cultivars, being maximum in ‘Frantoio’ and ‘Rotondella’ (59% and 55% reduction, respectively) and relatively lower in the other three varieties (-40%, -46% and − 44% in ‘Biancolilla’, ‘Caiazzana’, and ‘Salella’, respectively). A difference was not found in this parameter between inoculated and control plants in the other four cultivars (total shoot length of inoculated plants were significantly lower than control at DAI 53, but not at DAI 95).

Fig. 1
figure 1

Seasonal pattern of total shoot length per plant in (a) ‘Biancolilla’, (b) ‘Caiazzana’, (c) ’Frantoio’, (d) ‘Ogliarola’, (e) ‘Ortolana’, (f) ‘Racioppella’, (g) ‘Rotondella’, (h) ‘Salella’, and (i) ‘Tonda’ olive plants inoculated or non-inoculated (control) with Verticillium dahliae. Vertical bars indicate the standard error of the means. Separately for each cultivar and measuring date, one, two, and three asterisk/s indicate significant differences between inoculated and control plants according to one-way ANOVA at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively

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The total number of leaves per plant was significantly affected by the Time (T), Cultivar (CV), Inoculation (I), and the CV × T and T × I interactions (Table 1; Fig. 2). This parameter on average doubled during 95 days after inoculation. ‘Rotondella’ plants had the highest number of leaves per plant followed by ‘Biancolilla’, ‘Frantoio’, and ‘Tonda’, whereas this parameter was the lowest in ‘Ortolana’ and ‘Racioppella’ plants. On DAI 95, inoculated plants had on average of 8 leaves less than control (Table 1) with the highest decrease in ‘Caiazzana’ (-63%; -25 leaves/plant) (Fig. 2). No difference between inoculated and control plants was found in ‘Biancolilla’, ‘Ortolana’, ‘Racioppella’, ‘Salella’, and ‘Tonda’ (Fig. 2).

Fig. 2
figure 2

Seasonal pattern of total number of leaves per plant in (a) ‘Biancolilla’, (b) ‘Caiazzana’, (c) ’Frantoio’, (d) ‘Ogliarola’, (e) ‘Ortolana’, (f) ‘Racioppella’, (Boris Basile1*, Loredana Sigillo2, Alessandro Mataffo1, Giandomenico Corrado1

) ‘Rotondella’, (h) ‘Salella’, and (i) ‘Tonda’ olive plants inoculated or non-inoculated (control) with Verticillium dahliae. Vertical bars indicate the standard error of the means. Separately for each cultivar and measuring date, one, two, and three asterisk/s indicate significant differences between inoculated and control plants according to one-way ANOVA at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively

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Finally, main stem diameter was affected significantly only by Time (an average 18% increase between inoculation and DAI 95) and the CV × T interaction (Table 1), whereas this parameter was not affected by the Cultivar, the Inoculation treatment, and the other interactions. RGRSL, RGRNL and RGRStD largely varied among cultivars with RGRSL ranging between 0.021 and 0.060 cm/mm/day (‘Racioppella’ and ‘Rotondella’, respectively), RGRNL between 0.010 and 0.027 leaves/leaf/day (‘Ortolana’ and ‘Rotondella’, respectively); 0.0005 and 0.0045 mm/mm/day (‘Racioppella’ and ‘Rotondella’, respectively). The %ISL varied, respectively, between 31% (‘Ogliarola’) and 59% (‘Frantoio’), while %INL ranged between 24% (‘Biancolilla’) and 64% (‘Caiazzana’). The principal components analysis extracted five principal components (Table 2). Only the first two had an eigenvalue higher than 1 and together explained 76.2% of the total variance. The first principal component (Dim. 1) was positively correlated to RGRSL, RGRNL, RGRStD, and %ISL (Table 2). Conversely, the second principal component (Dim. 2) was positively correlated to %INL.

Table 2 Eigenvalue, percent of explained variance and correlation with the eight original variables of the six principal components (Dim.) extracted. RGRSL: relative growth rate for total shoot length in control plants; RGRNL: relative growth rate for total number of leaves in control plants; RGRStD: relative growth rate for stem diameter in control plants; %ISL: percent inhibition of the total shoot length per plant; %INL: percent inhibition of the total number of leaves per plant
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The bidimensional representation of the data done using the first two principal components allowed to separate the cultivars based simultaneously on the vegetative traits related to the growth vigor and on the susceptibility to the V. dahliae (Fig. 3). Cultivars located in the right two quadrants of the biplot (corresponding to positive values of Dim. 1) had a relatively more vigorous vegetative growth (highest RGRSL, RGRNL, and RGRStD) and high inhibition of shoot extension growth induced by V. dahliae inoculation (highest %ISL) (‘Frantoio’ and ‘Rotondella’ had a %ISL of 59% and 55%, respectively), whereas cultivars with the lowest scores of the first principal components had a relatively low vigour and %ISL (‘Ortolana’ and ‘Racioppella’ had a %ISL of 41% and 34%, respectively). The %INL was not related to the other variables included in the PCA, but this parameter clearly separated the cultivar ‘Caiazzana’, highlighting the slight defoliation that was found in the inoculated plants of this variety (Figs. 2b and 3).

Fig. 3
figure 3

Biplot of nine olive cultivars for the first two principal components extracted by the PCA. The vectors indicate the original variable loadings. RGRSL: relative growth rate for total shoot length in control plants; RGRNL: relative growth rate for total number of leaves in control plants; RGRSD: relative growth rate for stem diameter in control plants; Perc_I_SL: percent inhibition of the total shoot length per plant; Perc_I_NL: percent inhibition of the total number of leaves per plant

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3.2 Molecular diagnosis the plants and re-isolation of the pathogen

To verify the successful inoculation, two plants per treatment were analyzed with a diagnostic PCR assay at the end of the trial. After checking the identity of the fungal isolate before inoculation (Supplementary Fig. 1) we detected the fungus in the roots of only plants treated with the Verticillium, indicating that the plants of all the varieties were infected (Fig. 4). Moreover, vascular tissues from all inoculated plants appeared light brown, as expected, and the pathogen was consistently re-isolated on PDA medium.

Fig. 4
figure 4

An example of the diagnostic PCR of the plant’s roots at the end of the bioassay. 1: PCR negative control (water); 2: PCR positive control (Verticillium dahliae DNA); 3: control ‘Racioppella’; 4: infected Racioppella; 5: control ‘Caiazzana’; 6: infected ‘Caiazzana’; 7: control ‘Rotondella’; 8: infected ‘Rotondella’; 9: control ‘Frantoio’; 10: infected ‘Frantoio’; 11: control ‘Tonda’; 12: ‘infected ‘Tonda’

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4 Discussion

The phenotypic assessment of the plant response to a pathogen provides important guide to the selection of plant germplasm and it is often the first knowledge that can attract the interest of plant scientists and breeders. In this work, we evaluated the possible presence of resistant genotypes in a collection of olive varieties diffused in Southern Italy. Previous studies indicated that there is sufficient variability among the olive germplasm in the response to Verticillium wilt (Antoniou et al. 2008; Bubici and Cirulli 2012; Colella et al. 2008; García-Ruiz et al. 2014; López-Escudero et al. 2004; Martos-Moreno et al. 2006; Serrano et al. 2021). Moreover, it has been also proposed that germplasm adapted to specific and relatively limited geographical areas could be a possible source of resistance factors (Garcia-Ruiz et al. 2015), also considering that much of the highly tolerant cultivar are local (Garcia-Ruiz et al. 2015). These works more often focused on the defoliating pathotype although in the Mediterranean countries where Verticillium wilt is a common disease, the less severe non-defoliating type is more common (Markakis et al. 2009).

In our study, variability in disease expression was observed for two out of the three quantitative parameters related to vegetative growth under investigation. A distinction between more vulnerable cultivars can be made considering the inhibitory effect of the fungus on stem growth. The range of the responses was ample, with %ISL ranging between 31% and 59%. However, the quantitative analysis indicated that varieties that suffered less after inoculation were those with a limited vegetative growth. A similar distinction could be identified considering the number of leaves on the plants. The resistance to Verticillium in olive has been measured in different ways. Often it has been calculated considering the intensity of the disease in terms of the presence of symptomatic leaves (assessed in an ordinal scale) and more generally, taking into account the qualitative classification based on a mean disease score. Nonetheless, the ample variability of the olive germplasm also implies the need of a quantitative evaluation of the detrimental effect of the fungus, which should then incorporate the different vigor of the plants. Few works have previously investigated the influence on the vegetative growth expressed, for instance, in terms of biomass (Birem et al. 2016; Sanei and Razavi 2017). Our work is consistent with the lack of a single factor that can dichotomously distinguish the resistant and the susceptible varieties in olive, implying the absence of an inheritable locus of potential interest for plant breeding at least in our germplasm. As also discussed in the literature, a source for complete resistance to Verticillium wilt has not been yet found in olive, despite the analysis of several cultivars (Trapero et al. 2015; Chatzistathis et al. 2013). In olive, increased tolerance to Verticillium wilt is frequently associated with reduced disease progression (Trapero et al. 2013), which should also allow a more frequent recovery of the infected plants (López-Escudero et al. 2004). To avoid disturbance, we did not follow root growth and architecture, but it is likely that faster stem growth associated with an increased soil colonization. This difference among varieties may directly (e.g., amount of defense compounds) or indirectly (e.g., lignification) affect susceptibility to Verticillium infection and then the severity of the disease (Emmett et al. 2014).

Considering the mean values of the stem length, all the varieties were unable to grow consistently after the inoculation. This indicated not only the successful inoculation but also that none of the varieties was able to escape the infection. Nonetheless, the implication of this information should be moderated considering that we used favorable conditions for the fungal pathogen. It has been previously discussed that defense reactions to Verticillium seem to occur at a lower extent in susceptible varieties (Báidez et al. 2007; Bubici and Cirulli 2012). This indicates that the different severity of disease expression is probably multifactorial. A progeny-based screening for resistance to V. dahliae also indicated a highly variable response of the plants, with cases of transgressive phenotypes (Trapero et al. 2015).

The level of Verticillium infection in plants can vary greatly depending on various factors such as the variety, the pathogen strain, environmental conditions, and the plant’s genetic resistance (Pegg and Brady 2002; Klosterman et al. 2009; Fradin et al. 2009). In general, higher levels of infection can lead to more severe symptoms, such as wilting, yellowing of leaves, stunted growth, and in severe cases, plant death. However, it is important to note that the relationship between the level of infection and symptoms may not always be linear, also because symptomless olive trees can be infected. Symptoms may also depend on the stage of infection, as the pathogen can cause different symptoms at different stages of the plant’s life cycle. Moreover, the amount of Verticillium present in infected olive trees can depend on various factors such as the timing of infection, the pathogen’s ability to colonize the host, and the host’s ability to defend against the pathogen (López-Escudero et al. 2004; Erten and Yıldız 2011; Godena et al. 2022). Therefore, two olive trees with similar susceptibility to Verticillium may not necessarily have the same quantity of the pathogen in the infected tissues, also because different tissues/organs in the same plant can have different quantity of the fungus (Mercado-Blanco et al. 2003). For the olive-Verticillium interaction, the symptoms and severity of the infection not always correlated with the quantity of the pathogen present in a given tissue (Mercado-Blanco et al. 2003; Markakis et al. 2009).

Olive varieties are well known to have a large variability for several agronomic traits, which imposes caution when comparing the outcome of plant-pathogens interaction in genetically different material. Moreover, it has been previously shown that the disease parameters from experiments in natural environmental conditions are not well correlated to those in controlled conditions, especially for the identification of resistant genotypes (Serrano et al. 2021). Although the resistance to Verticillium under field conditions is determined by environmental and experimental factors (e.g., inoculum, pathotype, strain virulence, management practices), previous studies indicated that a growth reduction may be considered a proxy of the susceptibility level of the plants to V. dahliae (Sadras et al. 2000; Sanei and Razavi 2017). Our work highlighted the role of the different ability of the varieties in the shoot extension growth. Further studies will have to evaluate whether this feature influence the ability to recover from wilt disease symptoms, because the percentage of recovery significantly correlates with the resistance level in olive (López-Escudero and Blanco-López 2005).