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Article

The Induction of Resistance Against Verticillium Wilt of Olive by Previous Inoculation with a Low-Virulence Isolate of the Pathogen

by
Consuelo Martos-Moreno
1,
Antonio Santos-Rufo
1,2,*,
María del Carmen Raya-Ortega
1,
Francisco Javier López-Escudero
1 and
Miguel Angel Blanco-López
1
1
Excellence Unit ‘María de Maeztu’ 2020-23, Department of Agronomy, Campus de Rabanales, University of Cordoba, 14071 Cordoba, Spain
2
Department of Agroforestry Sciences, ETSI University of Huelva, 21007 Huelva, Spain
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(4), 420; https://doi.org/10.3390/horticulturae11040420
Submission received: 17 March 2025 / Revised: 12 April 2025 / Accepted: 14 April 2025 / Published: 15 April 2025
(This article belongs to the Special Issue Advances in Genetics, Breeding, and Quality Improvement of Olive)
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Abstract

:
Verticillium wilt of olive, caused by Verticillium dahliae Kleb., is a serious disease with no highly effective control methods currently available. Consequently, biological control strategies are being explored as viable and environmentally friendly alternatives. A natural recovery phenomenon has been observed in certain olive varieties following infection by low-virulence isolates of the pathogen, likely due to plant resistance mechanisms that may enhance defense against more virulent isolates. Based on these findings, a study was conducted to determine whether plants that had recovered from infection by a low-virulence isolate could exhibit increased resistance to highly virulent isolates. ‘Picual’ plants were first inoculated with a non-defoliating isolate, followed by inoculation with a defoliating isolate at different time intervals. The results demonstrate that prior infection with a non-defoliating isolate reduced disease severity caused by a defoliating isolate, particularly in susceptible cultivars like ‘Picual’. Treated plants exhibited slower disease progression and no mortality, whereas untreated plants developed severe symptoms and showed high mortality rates. A minimum interval of four months between inoculations with isolates of different virulence was crucial for achieving a significant reduction in disease severity. While this methodology has proven effective, further research is needed to elucidate the underlying mechanisms and identify additional biocontrol agents to enhance disease management strategies.

1. Introduction

The olive tree (Olea europaea L.) is the most widely cultivated oilseed woody crop globally, with over 95% of its cultivation area concentrated in the Mediterranean region, Southern Europe, North Africa, and the Middle East since the 1990s. In Spain, olive cultivation is particularly prominent in Andalusia, accounting for approximately 60% of the country’s total olive growing area [1,2]. Traditional propagation and cultivation methods have not significantly contributed to the loss of genetic diversity or environmental adaptation, likely due to factors such as low selection pressure from extensive farming practices and the strong geographical localization of olive varieties [1]. These characteristics have maintained a delicate balance between olive trees and their pathogens or other diverse agents, with serious outbreaks or losses occurring when this balance is disrupted by climatic conditions or by human intervention. The shift toward intensive olive growing has increased the vulnerability of this balance, potentially exacerbating existing issues or giving rise to new challenges.
In this context, Verticillium wilt of olive (VWO) has gained significant importance in recent decades. The spread of this disease, caused by the soil-borne fungus Verticillium dahliae Kleb., appears to be closely linked to the intensification of olive cultivation in soils already infested with the pathogen. This is particularly evident in areas where olive trees have been planted in soils previously used for crops such as cotton or vegetables, including tomato, pepper, potato, and eggplant [3,4,5,6,7].
VWO is a complex and difficult disease to control and requires integrated control strategies. Among the reasons that make its control difficult are the high survival rate of the pathogen in the soil and its location in the xylem of the plant (inaccessibility), the wide range of hosts in which it can survive, and the pathogenic variability of its populations with the existence of highly virulent pathotypes [3]. In Andalusia, the presence of the so-called defoliant pathotype (D) is notable. This is an extremely virulent population that causes green defoliation of the branches and death of the plant, unlike the less virulent non-defoliant pathotype (ND), which does not cause such defoliation. These D isolates, although originally (in the 1980s) found restricted to the Guadalquivir Marshes [8,9,10], have spread to other areas of Andalusia associated with cotton cultivation [3,11]. Therefore, this circumstance means that the disease has progressed from a potential risk to a real danger for olive plantations. The presence of the D isolate in commercial olive groves in Andalusia was described by López-Escudero and Blanco-López in 2001 [12], and currently these D isolates are the ones most frequently detected in the soils where olive trees are grown [3,5]. Likewise, the VWO problem may be even more serious if we take into account the high susceptibility of most of the cultivars used in Andalusia.
The use of resistant cultivars—or at least the avoidance of highly susceptible ones—is considered one of the most effective strategies for disease management within the framework of integrated control. Research to date indicates the existence of cultivars with moderate resistance to this pathogen [13,14]. Under controlled conditions, cultivars such as Empeltre, Frantoio, and Oblonga have shown resistance or moderate resistance to ND or D isolates of the fungus [15]. In contrast, other cultivars, including Picual—which is also used in the present study—are known to be more susceptible.
The methodologies for producing rooted cuttings [16] and evaluating resistance [17,18,19,20,21,22,23] are well established. López-Escudero et al. [19] initiated the evaluation of cultivars from the World Olive Germplasm Bank at CIFA “Alameda del Obispo” in Córdoba [24] using 9- to 12-month-old plants obtained through semi-hardwood cuttings under mist propagation. The ongoing evaluation of the varieties from this germplasm bank remains essential to identifying agronomically valuable genotypes with resistance to Verticillium wilt which could be used in areas prone to the disease and/or as a source of resistance [3,6,7].
An important aspect of the disease is that woody plants, such as olive trees, can recover after infection by low-virulence isolates, both under controlled conditions and in the field [25]. Some authors attribute this recovery to the compartmentalization or death of the pathogen as a result of new xylem formation [15,26]. This plant resistance mechanism could potentially confer a higher level of resistance against more virulent isolates. In this context, studying the interaction between both virulence populations of V. dahliae within the same olive plant is particularly relevant, as it could help evaluate the feasibility of an alternative biological control method through the application of less virulent V. dahliae isolates. Additionally, it may facilitate the search for new biocontrol agents, which have been scarce in this pathosystem.
This study aims to explore whether prior infection with non-defoliating isolates of V. dahliae can reduce the severity of VWO caused by defoliating isolates, potentially through mechanisms such as induced resistance or cross-protection.

2. Materials and Methods

2.1. Plant and Fungal Material

Nine-month-old rooted cuttings of the VWO-susceptible olive cultivar Picual were used as plant material. A total of 200 plants were obtained from Verticillium-free mother trees, sourced from the World Olive Germplasm Bank of CIFA “Alameda del Obispo” in Córdoba, Spain. The health of the mother trees was assessed through molecular diagnosis using real-time quantitative PCR (qPCR) with primers specific to V. dahliae [27]. Additionally, plant material was analyzed for other potential systemic pathogens using microbiological and molecular techniques. The material was thoroughly washed, and fragments containing phloem and xylem were collected and disinfected with sodium hypochlorite (0.5% Cl) for 1 min. After drying, these fragments were plated on Potato Dextrose Agar and incubated at 24 ± 3 °C for 7–14 days. The microbial species were identified both microbiologically and molecularly by gene amplification and sequencing using ITS1/ITS4 primers [28]. This procedure ensured that the cuttings were free from latent infections before being used in the experiment. The rooted softwood cuttings were acclimatized and grown in a greenhouse under misting conditions with a natural photoperiod, following the methodology outlined by Caballero and Del Río [29].
As fungal material, we used two isolates of V. dahliae: V4 (ND pathotype and race 1) and V117 (D pathotype and race 2). Both isolates belong to the mycotheca of the Agroforestry Plant Pathology Group (Department of Agronomy, María de Maeztu Unit of Excellence, University of Córdoba) and their characteristics have been fully established in previous works [7,8,30,31].

2.2. Inoculation (Production of Inoculum, Treatments, and Inoculation Schedule), Incubation Process, and Experimental Design

The technique for plant inoculation was based on the methodology of artificial inoculations reported by López-Escudero et al. [19]. The inoculum was prepared from single-spore cultures of V4 and V117 isolates, maintained in Plum Extract Agar (PEA) at 4 °C in the dark, that were transferred to Potato Dextrose Agar (PDA) dishes and incubated for 6 days at 24 °C in the dark. At the end of this time period, to obtain the inoculum, a sterile loop was slid over the surface of the plates after pouring 10–15 mL of sterile deionized water into each. The resulting conidial suspension was filtered through a double layer of sterile gauze and adjusted to a concentration of 107 conidia/mL using a Neubauer chamber.
The plants were extracted from their black plastic bags, with the root ball intact and washed first with pressurized water to remove the soil and a second time under tap water to loosen the fragments adhered to the roots, avoiding significant damage to the root system. After gently wounding the roots, mainly secondary ones, using disinfected scissors, the complete root system was submerged in the conidial suspension for 30 min (in containers with 4–5 L of inoculum); for the controls, sterile water was used instead of the inoculum. Finally, the plants were transplanted individually into sterile terracotta pots (16.5 cm in diameter and 15.5 cm in height) which contained sterile soil composed of sand, lime, and peat (1:1:1). In addition, for every 100 L of soil, the following were added: Superphosphate (225 g), NO3K (27.5 g), CO3Ca (15 g), SO4Mg (15 g), and CO3Mg (9 g). The plants were incubated in a controlled growth chamber at 22 ± 2 °C in the dark and at 95% RH for the first 3 days after inoculation to reduce, as much as possible, losses due to transplanting and the inoculation process. After this time, light and humidity were adjusted to a 14 h photoperiod (216 µEm−2 s−1 fluorescent light) and 80% HR. Each plant was watered with 150 mL of water according to its water needs, and 100 mL of a nutrient solution (Hakaphos green 15-10-15, 2 Mg, with Kifix-Mix microelements; BASF, Ludwigshafen, Germany) was added every 2 weeks.
The plants were inoculated with two isolates: V4 (non-defoliating, ND) and V117 (defoliating, D). The treatments consisted of the following: (i) inoculation with the V4 isolate (non-defoliating) alone, (ii) inoculation with the V117 isolate (defoliating) alone, used as an infection control, (iii) five sequential inoculation treatments, in which plants were first inoculated with V4 and subsequently challenged with V117 after intervals of 10 days, 1 month, 2 months, 4 months, or 6 months, (iv) five corresponding control groups for each time point mentioned above, in which plants were inoculated only with V4, with no subsequent V117 inoculation, and (v) a non-inoculated control group. The experimental inoculation process with V4 was carried out over 2 days due to the large amount of plant material and primarily to avoid inoculum transmission. On the first day, the control plants were processed, and on the second day, plants were inoculated with V4. In the case of double inoculation, plants previously inoculated with V4 (day 0) were later inoculated with V117 at intervals of 10 days, 1 month, 2 months, 4 months, or 6 months. Five Picual plants inoculated only with V117 served as infection controls. The inoculation schedule is detailed in Figure 1 and Table 1.
Table 1 summarizes the treatments used in the experiment. The following abbreviations were used: “C” for non-inoculated control plants, “ND” for plants inoculated with the non-defoliating isolate V4, and “D” for plants inoculated with the defoliating isolate V117. Combined treatments are indicated as ND/D (V4 followed by V117).
For the 10-day, 2-month, and 6-month intervals, four treatments were evaluated: C/C, ND/C, C/D, and ND/D. For the 1-month and 4-month intervals, only C/D and ND/D were included. These choices prioritized biologically relevant combinations while optimizing resources.
Plants were grouped and analyzed separately by time interval, and the experiment followed a randomized block design with 8–10 repetitions per treatment.

2.3. Evaluation of Disease Progress

Disease severity was assessed weekly from the second inoculation (not from the first inoculation) until week 17 post-inoculation. Disease symptoms were scored using a severity scale from 0 (healthy or asymptomatic plant) to 4 (dead plant), based on the percentage of plant tissue affected by chlorosis, leaf and bud necrosis, and/or defoliation [19]. The area under the disease progress curve (AUDPC) was calculated considering its percentage with regard to the maximum possible value that could be reached in the period of assessment, based on the formula from [32]: A U D P C = t / 2 × S 2 + 2 × S 3 + … + S i / 4 × n × 100 . Here, the term t represents the number of days between each observation. Si refers to the final mean severity at observation i, where 4 is the maximum disease rating. Finally, n denotes the number of observations made during the assessment period. Additionally, final mean severity (FMS) and percentage of dead plants (PDP) were also determined [33]. Representative photographs of symptom severity per treatment are available in the Supplementary Materials.
Plant infection was tested by the isolation of the fungus from affected shoots or leaf petioles of affected plants during the experiments. Shoots and branches of all dead plants were also tested for V. dahliae presence at the end of the experiment. Pieces of affected tissues were washed in running tap water, bark was removed, and woody tissue surfaces were disinfected in 0.5% sodium hypochlorite for 1 min. Chips of wood were placed onto PDA. Plates were incubated at 24 °C in the dark for 5–6 days.

2.4. Statistical Analysis

With the mean data of each plant, an analysis of variance (ANOVA) was performed to assess the differences in disease severity and plant mortality. The means were compared using Fisher’s Least Significant Protected Difference (LSD) test for multiple comparisons, with a significance level of 0.05. The analysis was performed using the General AOV/AOCV procedure from the Statistix 10 statistical package (Analytical Software, Version 8.1.1; Tallahassee, FL, USA).

3. Results

The defoliant isolates of V. dahliae caused a very severe response in ‘Picual’ plants not previously inoculated with the ND isolate. Thus, at the end of the evaluation period, mortality reached 100% among the plants and the AUDPC value obtained was 75.3%, corresponding to an extremely susceptible reaction (Figure 2). The response of the Picual cultivar varied depending on the time interval between inoculations and the treatment received (Table 2, Figure 3, Figures S1 and S2).
The progress curves of the average severity of ‘Picual’ reactions in each time interval (10 days, 1, 2, 4, and 6 months) are shown in Figure 3. Disease severity was evaluated approximately one month after the second inoculation, independent of the time interval between the two inoculations. In the intervals of 10 days, 2 months, and 6 months between inoculations with the ND/C treatment, the response of ‘Picual’ showed that the non-defoliant isolate (V4) caused light to moderate symptoms, with final mean severity values around 2 for the 10-day and 2-month intervals, and very mild symptoms (values below 1) in the 6-month interval (Table 2). The relatively low severity in the 6-month interval suggests a delayed response to the second inoculation with V117.
In relation to the comparison between the most important treatments, C/D and ND/D, for the lower time intervals (10 days, 1 and 2 months), there was an advance in the onset of the disease with the ND/D treatment with respect to its control C/D. The consistent development of symptoms in the protected ND/D treatment began 3 or 4 weeks after inoculation of the defoliant isolate, while in the C/D treatment, it occurred 7 weeks after inoculation with the defoliant isolate (Figure 3), but the severity of the reactions decreased in the protected treatment (ND/D) compared to the unprotected treatment (C/D) from intervals of 1 month (Table 2, Figure 3 and Figure S1).
The difference in reactions observed in ‘Picual’ between both treatments was more pronounced as the time interval between inoculations increased, the difference being especially evident at 4 and 6 months (Table 2, Figure 3 and Figure S2). For these time intervals, the plants corresponding to the ND/D protected treatment showed a slow development of the disease that corresponded to final average severity values of around 1.5 (light–moderate) and plant mortality of 0% compared to the severity values of very severe symptoms (close to the death of the plant, final severity value ≥ 3.5) and very high mortality (≥75%), observed in the plants with the C/D treatment (Table 2).
The analysis of variance for each time interval showed that the AUDPC was significantly higher for ND/D treatment in the 10-day and 2-month intervals and the same for the 1-month interval between the two inoculations. However, when the time interval was greater than or equal to 4 months, the ND/D treatment reduced the AUDPC value in relation to the C/D control treatment (Table 3). When the interval was 6 months, the AUDPC values of the ND/D treatment did not differ significantly from those shown by the ND/T treatment (Table 3).
Once the evaluation period was over, the plants remained in the controlled environment culture chamber for a variable period according to the time interval between treatments (Table 4).
At 16 weeks after inoculation with the defoliant isolate, regardless of the time interval, practically all the plants belonging to the C/D treatment were dead (Table 4). On the contrary, those belonging to the ND/D treatment showed a reduction in the disease that depended on the time interval between inoculation with the non-defoliating and defoliating isolates of V. dahliae. Thus, for the 10-day interval, mortality due to the defoliant isolate varied from 40% at 12 weeks (Table 2) to 80% at 16 weeks (Table 4). In the rest of the intervals studied, mortality in the plants of this treatment did not vary after 12 days with the defoliant isolate. In general, the disease in the plants under this treatment (ND/D) began to subside, with better recovery observed in plants in which the inoculation with the defoliant isolate was carried out 2 months after the previous inoculation with the least virulent (non-defoliating) isolate of the pathogen (Table 4, Figure 4).

4. Discussion

At present, VWO is considered one of the most serious and significant diseases affecting this crop. Studies on this disease have demonstrated its complexity and emphasized the need for continued research into control measures that reduce the level of pathogen inoculums in the soil, limit its ability to infect and colonize the plant, and consequently mitigate the disease.
Within the framework of integrated disease control, biological control measures are currently considered viable, effective, and environmentally friendly alternatives. In this context, various interactions associated with different biological control mechanisms for VWO have been identified. Many of these interactions have been studied in recent years by the Agroforestry Pathology Group AGR-216 of the University of Córdoba, in collaboration with research groups from the IAS_CSIC [7,35].
Among them, investigations about the interaction between V. dahliae isolates of varied virulence on olive cultivars showed that previous infection with a non-defoliating isolate was effective in reducing the disease caused by a defoliating isolate. This reduction was greater in cultivars susceptible to the defoliant isolate. Thus, plants of the Picual cultivar corresponding to the protected treatment (ND/D) showed a slower development of the disease compared to the unprotected treatment (C/D). At 12 weeks after inoculation, the defoliant isolate caused very severe symptoms and 83.3% of dead plants in the non-protected treatment (C/D); on the contrary, with the protected treatment (ND/D), the plants showed moderate reactions and 0% death.
In this experiment, the time interval between the two inoculations (ND and D) was at least 10 days and the plants recovered from the infection by the non-defoliating isolate. This recovery phenomenon has been primarily attributed to the inactivation or restriction of the fungus within the xylem, driven by mechanisms in the plant that are not yet fully understood. These mechanisms may potentially enhance the plant’s resistance to more virulent isolates of the pathogen. Studies have demonstrated that Verticillium dahliae can persist in asymptomatic plants for varying periods, depending on environmental conditions [17,36]. This latent presence of the fungus suggests a dynamic interaction between the pathogen and the host, where the plant may suppress fungal activity without completely eliminating it, potentially contributing to a form of tolerance or partial resistance. Further research is needed to elucidate the underlying mechanisms and their implications for disease management.
The effect of different time intervals between inoculations with isolates of varying virulence (non-defoliating, ND, and defoliating, D) on disease reduction has been studied in the ‘Picual’ olive cultivar. When considering the AUDPC (the area under the disease progress curve) value as the primary parameter to evaluate disease progression, the results indicate that a minimum interval of 4 months between inoculation with the non-defoliating isolate and subsequent infection with the defoliating isolate is required to achieve a significant reduction in disease severity. However, other parameters, such as final disease severity and the percentage of dead plants, suggest that even 1 month after inoculation with the non-defoliant isolate protected plants (ND/D) exhibit a slightly less susceptible response compared to unprotected plants (C/D). This implies that early inoculation with the non-defoliating isolate may initiate protective mechanisms in the plant, although a longer interval is necessary for more pronounced disease reduction. These findings highlight the potential of using non-defoliating isolates to induce resistance or tolerance against more aggressive defoliating strains of Verticillium dahliae.
At shorter time intervals (10 days, 1 month, and 2 months) between inoculations, the ND/D treatment (pre-inoculation with an ND isolate followed by a D isolate) resulted in earlier onset of disease symptoms compared to the C/D treatment (control plants inoculated only with the D isolate). In these cases, the final average severity in unprotected plants (C/D) indicated that the D isolate caused severe or very severe symptoms when the interval between inoculations was 2 months or less. This suggests that shorter intervals between ND and D inoculations did not provide sufficient time for the plant to develop protective mechanisms against the more aggressive D isolate. However, when the time interval between inoculations with the ND and defoliating (D) isolates was extended to 4 or 6 months, a different pattern emerged. In these cases, plants treated with ND/D exhibited slower progression of the disease, with final severity values ranging from light to moderate and no plant mortality (0%). In contrast, plants in the C/D treatment showed very high symptom severity and mortality rates (≥75%). This indicates that longer intervals between inoculations allowed the plant to develop a more effective defense response, potentially through mechanisms such as induced resistance or cross-protection, which mitigated the impact of the D isolate. These findings highlight the importance of the time interval between inoculations in determining the effectiveness of prior exposure to a non-defoliating isolate in reducing the severity of Verticillium wilt caused by a defoliating isolate. A minimum interval of 4 months appears to be necessary for the plant to establish a robust protective response, significantly reducing disease severity and mortality. From a practical standpoint, this suggests that inoculation with the non-defoliating isolate should ideally take place during the early developmental stages, such as in the nursery phase, to ensure sufficient time for resistance induction before transplantation into infested soils. This approach, while requiring further validation under commercial conditions, could be integrated into sustainable disease management strategies in olive cultivation.
The methodology used in these experiments has successfully reduced the disease in plants protected with the ND isolate. However, it has not allowed us to identify the mechanisms involved in this reduction, as reported in other studies on induced resistance [37,38], and they remain unknown. There are few references in the consulted literature regarding biological control of Verticillium wilt in olive trees [39,40,41,42]. Various biological control mechanisms could be involved [43], including competition for nutrients or infection sites, protection, and induced resistance, as described in other pathosystems [44,45]. The results of this study represent an initial approach to the biological control of this disease and highlight the need for further research to elucidate the mechanisms involved. Additionally, they emphasize the importance of exploring new biocontrol agents, such as non-pathogenic isolates of V. dahliae in olive trees, other Verticillium species, or even organisms from different genera.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11040420/s1: Figure S1. Reactions of ‘Picual’ according to different treatments and time intervals between inoculations. Figure S2. Reactions of ‘Picual’ according to different treatments and time intervals between inoculations.

Author Contributions

Data analyses, writing, and editing of the manuscript: C.M.-M., A.S.-R., M.d.C.R.-O., F.J.L.-E. and M.A.B.-L.; data analyses and reviewing of the manuscript: A.S.-R. and F.J.L.-E.; conception and design of the study, acquiring funding, providing supervision, and revising of the manuscript: F.J.L.-E. and M.A.B.-L. All authors have read and agreed to the published version of the manuscript.

Funding

This research work was supported by Spaińs Comisión Interministerial de Ciencia y Tecnología (Project AGF-97-0546) and by the European Commission (Project QLRT-1999-1523).

Data Availability Statement

All data analyzed in this study are included in this article.

Acknowledgments

We want to thank to people of Research Group AGR-216 from the Department of Agronomy of University of Córdoba (Unit of Excellence Maria de Maeztu), and particularly Francisca Luque Marquez for their technical help in the lab and Antonio Trapero Casas for reviewing the statistical analyses.

Conflicts of Interest

The authors declare no conflicts of interest.

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Horticulturae 11 00420 g001
Figure 1. A schematic representation of the approximate time scale of the process of sequential inoculation of plants of the Picual cultivar with the two isolates of Verticillium dahliae: V4 (ND) and V117 (D).
Figure 1. A schematic representation of the approximate time scale of the process of sequential inoculation of plants of the Picual cultivar with the two isolates of Verticillium dahliae: V4 (ND) and V117 (D).
Horticulturae 11 00420 g001
Horticulturae 11 00420 g002
Figure 2. Disease progression in the Picual cultivar inoculated with the defoliant isolate of Verticillium dahliae. Vertical bars represent the standard deviation of the mean.
Figure 2. Disease progression in the Picual cultivar inoculated with the defoliant isolate of Verticillium dahliae. Vertical bars represent the standard deviation of the mean.
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Horticulturae 11 00420 g003
Figure 3. The progress of the severity of the reactions of the Picual cultivar at different time intervals between inoculation treatments with Verticillium dahliae. Vertical bars represent the standard error of the mean.
Figure 3. The progress of the severity of the reactions of the Picual cultivar at different time intervals between inoculation treatments with Verticillium dahliae. Vertical bars represent the standard error of the mean.
Horticulturae 11 00420 g003
Horticulturae 11 00420 g004
Figure 4. The evolution of the disease in the Picual cultivar for the intervals of 2 or 4 months between previous inoculation with the non-defoliant isolate and inoculation with the defoliant isolate of Verticillium dahliae. Vertical bars represent the standard error of the mean.
Figure 4. The evolution of the disease in the Picual cultivar for the intervals of 2 or 4 months between previous inoculation with the non-defoliant isolate and inoculation with the defoliant isolate of Verticillium dahliae. Vertical bars represent the standard error of the mean.
Horticulturae 11 00420 g004
Table 1. Treatments studied in experiment regarding sequence of inoculation.
Table 1. Treatments studied in experiment regarding sequence of inoculation.
Treatment xSequence of Inoculation
First InoculationSecond Inoculation
C/CSterile waterSterile water
C/DSterile waterDefoliating pathotype
ND/CInductorSterile water
ND/DInductorDefoliating pathotype
D-Defoliating pathotype
x “C” refers to non-inoculated control plants; “ND” refers to plants inoculated with the non-defoliating isolate V4; and “D” refers to plants inoculated with the defoliating isolate V117. The treatment combinations are as follows: C/C: control at both time points; ND/C: inoculated with V4 at the first time point, control at the second; C/D: control at the first time point, inoculated with V117 at the second; ND/D: inoculated with V4 at the first time point, followed by inoculation with V117 at the second. These treatments were applied at different intervals: 10 days, 1 month, 2 months, 4 months, and 6 months.
Table 2. Mean severity of reactions and percentage of dead plants of Picual cultivar for each treatment and time interval between inoculations x.
Table 2. Mean severity of reactions and percentage of dead plants of Picual cultivar for each treatment and time interval between inoculations x.
Time Interval (Months)TreatmentMean SeverityDead Plants (%)
10 daysC/D2.6 b10
ND/D3.6 a40
ND/C1.8 b0
1C/D3.9 a80
ND/D3.0 a50
2C/D3.6 a40
ND/D2.3 b20
ND/C1.5 b10
4C/D3.9 a87.5
ND/D1.7 b0
6C/D3.9 a75
ND/D1.6 b0
ND/C0.7 c0
x Control plants (C) or those inoculated with the non-defoliant isolate (ND) were subsequently inoculated with a defoliant isolate of V. dahliae (C/D, ND/D) at different time intervals. For each time interval, the figures in columns followed by different letters differ significantly at a probability level of p = 0.05 according to Fisher’s protected DMS contrast [32]. The values represent the mean of 8–10 repetitions per treatment. Standard errors were consistently below 0.4 for mean severity and below 10% for mortality percentages.
Table 3. The area under the disease progression curve in ‘Picual’ for each treatment and time interval between inoculations x.
Table 3. The area under the disease progression curve in ‘Picual’ for each treatment and time interval between inoculations x.
TreatmentTime Interval Between Inoculations y
10 Days1246
C/D24.1 b37.5 a33.7 b58.0 a49.6 a
ND/D54.5 a49.4 a58.7 a29.2 b30.3 b
ND/C26.6 b 40.7 ab 27.9 b
x Control plants (C) or those inoculated with the non-defoliant isolate (ND) were subsequently inoculated with a defoliant isolate of V. dahliae (C/D, ND/D) at different time intervals. y For each time interval, different letters in the same column indicate significant differences between values at a probability level of p = 0.05 according to Fisher’s protected DMS contrast [34].
Table 4. The evolution of the severity of the reactions and the percentage of dead plants of ‘Picual’ for each treatment and time interval between inoculation of the non-defoliant and defoliant isolate of Verticillium dahliae x.
Table 4. The evolution of the severity of the reactions and the percentage of dead plants of ‘Picual’ for each treatment and time interval between inoculation of the non-defoliant and defoliant isolate of Verticillium dahliae x.
Interval (Months)Weeks After Inoculation with Isolate D Mean SeverityDead Plants (%)
C/DND/DC/DND/D
10 days143.23.62060
163.73.77080
383.83.59080
1134.03.110050
144.03.210050
154.03.210050
164.03.210060
344.02.610060
2143.92.39020
153.92.29020
194.02.210020
214.02.210020
254.01.810020
304.01.410020
4133.91.787.50
164.01.51000
204.01.21000
214.01.11000
234.01.11000
254.01.01000
6143.91.787.50
164.01.91000
x Control plants (C) or those inoculated with the non-defoliant isolate (ND) were subsequently inoculated with a defoliant isolate of V. dahliae (C/D, ND/D) at different time intervals. The values represent the mean of 8–10 repetitions per treatment. Standard errors were consistently below 0.3 for mean severity and below 12% for mortality percentages across all time points.
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Martos-Moreno, C.; Santos-Rufo, A.; Raya-Ortega, M.d.C.; López-Escudero, F.J.; Blanco-López, M.A. The Induction of Resistance Against Verticillium Wilt of Olive by Previous Inoculation with a Low-Virulence Isolate of the Pathogen. Horticulturae 2025, 11, 420. https://doi.org/10.3390/horticulturae11040420

AMA Style

Martos-Moreno C, Santos-Rufo A, Raya-Ortega MdC, López-Escudero FJ, Blanco-López MA. The Induction of Resistance Against Verticillium Wilt of Olive by Previous Inoculation with a Low-Virulence Isolate of the Pathogen. Horticulturae. 2025; 11(4):420. https://doi.org/10.3390/horticulturae11040420

Chicago/Turabian Style

Martos-Moreno, Consuelo, Antonio Santos-Rufo, María del Carmen Raya-Ortega, Francisco Javier López-Escudero, and Miguel Angel Blanco-López. 2025. "The Induction of Resistance Against Verticillium Wilt of Olive by Previous Inoculation with a Low-Virulence Isolate of the Pathogen" Horticulturae 11, no. 4: 420. https://doi.org/10.3390/horticulturae11040420

APA Style

Martos-Moreno, C., Santos-Rufo, A., Raya-Ortega, M. d. C., López-Escudero, F. J., & Blanco-López, M. A. (2025). The Induction of Resistance Against Verticillium Wilt of Olive by Previous Inoculation with a Low-Virulence Isolate of the Pathogen. Horticulturae, 11(4), 420. https://doi.org/10.3390/horticulturae11040420

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