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Trichoderma species are known for their ability to control many plant diseases. In this study, fifteen (T1–T15) indigenous strains of Trichoderma spp. isolated from rhizosphere soils of olive in northern Algeria were investigated for their biocontrol potential against eleven strains of Verticillium dahliae, the causal agent of wilting on olive (Olea europea cv. europea L.). Direct and indirect confrontation assays were performed to investigate if isolates of Trichoderma affect mycelium growth. In addition, gas chromatography–mass spectroscopy (GC–MS) was applied to identify the volatile organic compounds (VOCS) produced by the T2 isolate. Besides, Trichoderma isolates were screened for the production of extracellular enzymes on solid medium including chitinases, proteasesand cellulases. Results showed that Trichoderma isolates demonstrated an effective potential in reducing mycelium growth against V. dahliae in vitro. T12 was the highest antagonistic of the Trichoderma isolates by direct confrontation assay method. This isolate exhibited an average inhibition rate of 68.86% against V. dahliae isolates, whereas the evaluation of volatile metabolites effect revealed that inhibition percentage of radial growth varies between 55.26 and 65.93%. Importantly, results showed the presence of 33 compounds detected in the methanolic and hexanic fraction of T2 isolate, including important volatile compounds with antifungal activities such as Undecane, Octadecane, Eicosane, 13-Docosenamide, (Z), Hexadecanamide, 9-Octadecenamide, (Z), Cyclopentanone, 2-methyl, Tetradecanoic acid, propyl ester, Tetradecanoic acid, oleic acid, and n-Hexadecanoic acid. Interestingly, all isolates tested showed chitinolytic, proteolytic and cellulolytic activity. T2 and T12 were the effective Trichoderma isolates showing high chitinase production on solid medium. Besides that, isolates T2 and T6 demonstrated the highest chitinolytic and proteolytic activity with values 0.043 µmol/ml/min and 0.019 µmol/ml/min, respectively. Overall, the isolates, T2, T6, and T12 are the most effective agents against olive wilt pathogen V. dahliae in vitro. Hence, they could be further investigated for biocontrol of olive wilt in vivo.

Verticillium wilt of olive, caused by Verticillium dahliae Kleb., is one of the most important diseases affecting olive crops in the Mediterranean area. With the aim to evaluate the role of Phloeotribus scarabaeoides (Bernard) (olive bark beetle) as a dispersal vector of V. dahliae, several experiments were conducted in semi-controlled conditions from May 2009 to April 2012. Groups of olive trees (2.5-year-old) certified free from V. dahliae were covered by a mosquito net and exposed to adults of P. scarabaeoides by three different ways: (1) branches or trunks collected in several olive orchards from trees severely affected by Verticillium wilt and showing apparent entry holes (mating galleries) of P. scarabaeoides, (2) adults of olive bark beetle extracted from damaged branches collected in the field, (3) adults from damaged branches that were superficially inoculated with V. dahliae. The fungus V. dahliae was not detected either by microbiological and molecular techniques from shoots of olive trees with galleries of the insect or from any of the tissues of the collected beetle adults from the galleries. Additionally, Verticillium wilt disease symptoms were not observed in olive trees exposed to the olive bark beetles. Moreover, the pathogen was never detected from any beetle adults that were recovered from the mating galleries of branches or trunks collected in several olive orchards from trees severely affected by Verticillium wilt. We conclude that P. scarabaeoides is not a vector of V. dahliae under the investigated experimental conditions.

Verticillium wilt, caused by Verticillium dahliae, challenges olive cultivation and an Integrated Disease Management (IDM) approach is the best-suited tool to combat it. Since 1998, an IDM strategy in an orchard (called Granon, Spain) of the susceptible cv. Picual was conducted by increasing planting density with moderately resistant cv. Frantoio, chemical weed control, and replanting of dead olives with cv. Frantoio following soil solarization. The Verticillium wilt epidemic in Granon orchard was compared to the epidemic in a non-IDM orchard (called Ancla, Spain) with plowed soil and dead ‘Picual’ olives replanted with the same cultivar. Field evaluations (2012-2013) showed an incidence and severity of the disease as Picual-Ancla > Picual-Granon > Frantoio-Granon. The spatiotemporal dynamics of the Verticillium epidemics from 1998 to 2010 were monitored with digital images using SIG. The annual tree mortalities were 5.6% for ‘Picual’ olives in Ancla orchard, and 3.1 and 0.7% for ‘Picual’ and ‘Frantoio’ olives in Granon orchard, respectively. There was a negative relationship between the mortality of olive trees (%) by the pathogen and the height (m) above sea level. The annual mortality of cv. Picual olives was positively correlated with spring rainfalls. The Index of Dispersion and Beta-Binomial distribution showed aggregation of Verticillium-dead olives. In conclusion, this IDM strategy considerably reduced the disease in comparison with traditional agronomic practices.

The use of genetic resistance is likely the most efficient, economically convenient and environmentally friendly control method for plant diseases, as well as a fundamental piece in an integrated management strategy. This is particularly important for woody crops affected by diseases in which mainly horizontal resistance mechanisms are operative, such as Verticillium wilt, caused by Verticillium dahliae. In this study, we analyzed the variability in resistance to Verticillium wilt of olive trees in progenies from five crosses: ‘Picual’ × ‘Frantoio’, ‘Arbosana’ × ‘Koroneiki’, ‘Sikitita’ × ‘Arbosana’, ‘Arbosana’ × ‘Frantoio’ and ‘Arbosana’ × ‘Arbequina’ and their respective reciprocal crosses. Additionally, seedlings of ‘Picual’ and ‘Frantoio’ in open pollination were used as controls. In October 2016 and 2018, the fruits were harvested, and seeds germinated. Six-week-old seedlings were inoculated by dipping their bare roots in a conidial suspension of V. dahliae, and disease progress in terms of symptom severity and mortality was evaluated weekly. Additionally, seedling growth was evaluated every two weeks. At the end of the experiment, no significant differences were found for any of the assessed parameters when reciprocal crosses were compared. These results suggest that there is no maternal or paternal effect in regard to the heritability of resistance. In addition, this study identifies the best crosses for obtaining the highest number of resistant genotypes, highlighting the importance of the selection of specific cultivars to optimize the breeding process.

Fungal viruses, also known as mycoviruses, are widespread in all major groups of fungi. Mycoviruses from plant pathogens can reduce the virulence of their host fungus and have therefore potential as biological control agents. This has spurred the identification of novel mycoviruses in plant pathogens, research which is greatly contributing to our understanding of these organisms. In this work, we report the characterization of a novel monopartite mycovirus from Verticillium dahliae, the main causal agent of Verticillium wilt. This novel mycovirus, which we termed Verticillium dahliae RNA virus 1 (VdRV1), was identified in three different isolates of V. dahliae collected in olive growing areas of the Guadalquivir valley, southern Spain. We determined that the VdRV1 genome is a positive (+) single-stranded (ss) RNA, 2631 nucleotides in length, containing two open reading frames. VdRV1 showed few similarities with known mycoviruses, only with a group of unassigned (+) ssRNA mycoviruses which are related to plant viruses classified within the family Tombusviridae. However, phylogenetic analysis revealed that VdRV1 and the unassigned (+) ssRNA mycoviruses have a closer relationship with recently reported invertebrate viruses. This result indicates that as more viral sequences become available, the relationships of mycoviruses with viruses from other hosts should be reexamined. Additionally, the work supports the hypothesis of a heterogeneous origin for mycoviruses.

Verticillium wilt in olive is considered the most serious biotic threat to this crop. Effective control of this disease relies on an integrated disease management strategy in which the biological control has an important role nowadays. This work describes the potential effect of 44 plant extracts and 20 essential oils against Verticillium dahliae. The results demonstrate the in vitro and in planta effectiveness of essential oil from Thymus, in particular Thymus sp. 04 (prepared in the laboratory), and the commercial product Thymus sp. 01, against Verticillium dahliae. The inhibition of mycelial growth and microsclerotia reached 100% in both treatments and achieved a disease reduction in olive plants by 65% and 42% for Thymus sp. 04 and sp. 01, respectively. These treatments showed the potential for essential oils use in the control of this pathogen in the frame of an integrated disease management strategy. This is the first report of the use of essential oils to control Verticillium wilt in olive plants. Further studies are warranted to identify the bioactive compounds in the essential oil that control V. dahliae and evaluate their potential use as natural fungicides.

Four soil solarization experiments were completed in three commercial olive orchards infested with Verticillium dahliae in southern Spain. Three of the experiments used lines of trees and one used individual plants. Plantations had different initial inoculum densities of the pathogen. Initial studies indicated that highly virulent (cotton-defoliating) isolates of the pathogen were present in Marinaleda (experiment I), which represents the first record of such isolates affecting olive trees in Europe. Solarization treatments were applied to lines of trees for either one (single) or two consecutive (double) years. Solarization significantly reduced pathogen populations in the top 20 cm of soil for at least 3 years in relation to control plots. Pathogen reduction after the single solarization obscured effects of the second solarization treatment. Decrease of inoculum density in soil by solarization did not correspond to a similar reduction in disease severity. Disease severity was reduced only in orchards with medium or high initial inoculum densities. A second soil solarization treatment did not improve the effect of single solarization on Verticillium wilt control. In orchards with low inoculum densities, soil solarization did not result in significant differences in disease incidence and severity, but improved recovery of trees from the disease. Soil-solarized plots remained free of weeds, but tress in solarized plots did not show significant growth increase measured by trunk perimeter.

Pistachio (Pistacia vera L.) trees in the Castilla La Mancha and Andalusia regions of central and southern Spain are grown close to olive orchards, which are often severely affected by Verticillium dahliae. During the last decade, wilt and death of one or several branches have been observed on pistachio (cv. Kerman) scions grafted on rootstock (P. terebinthus). Discoloration of vascular tissue was occasionally observed. In five surveyed orchards, incidence of affected trees was less than 2%. Wood chips with the bark removed from symptomatic trees were washed in running tap water, surface disinfested in 0.5% sodium hypochlorite for 1 min, and placed onto potato dextrose agar plates incubated at 25°C in the dark. Isolates were identified as V. dahliae on the basis of the characteristics of microsclerotia, conidiophores, and conidia. V. dahliae isolate V117 from olive was used as reference (1). The fungus was also isolated from soil in pistachio orchards using wet sieving and a modified sodium polypectate agar medium (1). Inoculum density varied from 0 to 4.73 microsclerotia per gram of soil. P. terebinthus seedlings were inoculated with two isolates of V. dahliae from pistachio trees by injecting the stems with 50 μl of a conidial suspension (107 conidia per ml) (2). Wilt symptoms of varying severity developed in 12 and 15 seedlings of the 20 pistachio seedlings inoculated with each of two isolates. No symptoms developed on the control seedlings. The pathogen was recovered from stem tissues of inoculated plants. To our knowledge, this is the first report of Verticillium wilt of pistachio in Spain. This study demonstrates the susceptibility of certain rootstocks to V. dahliae and the importance of using resistant rootstocks, such as UCBI (2), in pistachio plantations established on soils infested by V. dahliae. References: (1) F. J. López-Escudero and M. A. Blanco-López. Plant Dis. 91:1372, 2007. (2) D. P. Morgan et al. Plant Dis. 76:310, 1992.

Verticillium wilts caused by Verticillium spp. are among the most challenging plant diseases to control and affect numerous hosts worldwide. Due to the lack of effective, conventional control methods, integrated control strategies provide a promising approach to manage these diseases. The non-pathogenic Fusarium oxysporum strain FO12 was reported in previous studies to be an effective biocontrol agent against Verticillium dahliae, however, its mode of action remains to be elucidated. In this study, complementary in vitro and in vivo experiments were conducted in order to explore the implications of inhibitory substances and rhizosphere competence in antagonistic effects of FO12 against V. dahliae and V. longisporum. Volatile organic compounds and soluble substances produced by FO12, which caused significant inhibition of mycelial growth and microsclerotia viability in the two tested Verticillium species, were identified by means of gas and liquid chromatography-mass spectrometry. We showed that the antagonistic effect of F. oxysporum FO12 is partially due to the production of bioactive compounds such as 3-methyl-1-butanol and 2-methyl-1-butanol, among others. Several metabolic pathways of FO12 were altered upon contact with V. dahliae ELV22 volatiles. The reduced production of alpha, alpha-trehalose, a metabolite used in starch and sucrose metabolism, suggests that the biocontrol agent activates its stress response in the presence of the phytopathogen. Microscopic analysis using sGFP-tagged FO12 on oil seed rape as a model plant suggests that the biocontrol strain is an efficient root colonizer, which could compete with V. dahliae in the same ecological niche. The findings obtained in this study provide new insights into the mode of action of this potential biocontrol agent, which are relevant for controlling Verticillium wilt through an ecologically friendly approach.

Purpose: Water consumption and vegetative growth in resistant (“Frantoio”) and susceptible (“Picual”) olive cultivars infected with Verticillium dahliae were assessed. Methods: Plants were inoculated with the pathogen and disease evaluated in growth chamber. Mean daily water consumption and vegetative growth were recorded during 13 weeks. Results: Water consumption significantly decreased in inoculated plants compared to non-inoculated plants during the 3rd - 6th weeks after inoculation period. From the 9th week, a significantly lower consumption occurred in infected “Picual” plants compared with “Frantoio” plants. Vegetative growth was reduced in inoculated plants of both cultivars, although the reduction of the fresh weight was remarkably higher in “Picual” compared with controls due to defoliation and tissue desiccation, impeding the production of new shoots and leaves. Conversely, in “Frantoio”, defoliation was significantly reduced to low levels. Conclusions: Water consumption and vegetative growth in plants of resistant (“Frantoio”) and susceptible (“Picual”) olive cultivars inoculated by Verticillium dahliae were consistently associated with the progress of the wilt symptoms and the level of resistance of the cultivar. Growth and water consumption reductions in “Picual” were irreversible in almost all plants, while “Frantoio” plants were able to maintain water translocation, overcome the infection and avoid vascular occlusion, and restart vegetative growth.

Verticillium wilt of olive (VWO) caused by Verticillium dahliae is considered one of the most serious diseases in olives. Effective control of this disease requires an integrated management strategy in which implementing biological control is a potential tool. Thus, the aim of this study was to evaluate the effect of 16 natural biological compounds, including biological control agents, organic amendments and one essential oil, under field conditions. The compounds were selected out of 220 treatments previously evaluated against V. dahliae under controlled conditions in both in vitro and in planta experiments. Three experiments were conducted under three different scenarios for the natural infection of V. dahliae in the Andalusian Region (southern Spain): under semi-controlled conditions on potted plants grown with naturally infested soil (Experiment I); new plantation in a field with high inoculum density (ID) of V. dahliae in the soil (Experiment II); replanting a commercial olive orchard affected by Verticillium wilt with 1 or 30 year-old trees (Experiment III). In the Experiment I, the non-pathogenic strain of Fusarium oxysporum (FO12) and a commercial essential oil from Thymus sp. were the most effective treatments, achieving a total reduction of the ID two months after planting. In Experiment II, the grape marc compost (CGR03) was the most effective treatment in reducing the ID of the pathogen in soil up to 100%. In Experiment III, both FO12 and CGR03 treatments reduced significantly the disease incidence in both 1 or 30 year-old olive plants in comparison with the untreated control plants (P = 0.05). This work provides a practical basis for the use of these selected treatments and a useful methodology for the establishment of future field experiments for research on the biological control of VWO.

The non-pathogenic Fusarium oxysporum FO12 strain isolated from cork (Quercus suber) was one of the most effective treatments against Verticillium dahliae from among more than 200 natural compounds evaluated in previous studies. Based on the results of previous work, the present study aims to determine the potential of FO12 as a biological control agent against Verticillium wilt of olive (VWO). To this end, several in vitro and in vivo experiments were carried out to evaluate the effectiveness of FO12 against V. dahliae. Dual cultures were conducted, showing a significant reduction in the mycelial growth of V. dahliae. However, inhibition zones were not observed in vitro. Four different culture fractions of FO12 were prepared to evaluate their effect on the reduction of viable propagules of V. dahliae in soil and on the disease progression in inoculated plants. Treatments with crude culture broth of FO12 resulted in a total reduction of the inoculum density of V. dahliae in naturally infested soils as well as in the progression of VWO under controlled conditions. Treatments with conidial suspension or chlamydospores were also effective in reducing the inoculum density in the soil and the disease severity in olive plants. This work provides useful information about the efficacy of several FO12-based culture fractions against VWO under controlled conditions, which could be the basis for the development of effective commercial formulations in the coming future.

This study investigated starch content, amount of pathogen DNA and density of occluded vessels in healthy and Verticillium dahliae infected olive shoots and stems. Starch hydrolysis is considered a mechanism to refill xylem vessels that suffered cavitation by either, drought conditions or pathogen infections. The main objective of this work was to evaluate this mechanism in olive plants subjected to V. dahliae infection or to drought conditions, in order to know the importance of cavitation in the development of wilting symptoms. In initial experiments starch content in the shoots was studied in trees of cultivars differing in the level of resistance growing in fields naturally infested with V. dahliae. The starch content, esteemed by microscopic observation of stem transversal sections stained with lugol, decreased with the level of symptom severity. Results were confirmed in a new experiment developed with young plants of cultivars 'Picual' (highly susceptible), 'Arbequina' (moderately susceptible) and 'Frantoio' (resistant), growing in pots under greenhouse conditions, either inoculated or not with V. dahliae. In this experiment, the pathogen DNA content, quantified by real-time PCR, and the density of occluded vessels, recorded by microscopic observations of transversal sections stained with toluidine blue, were related to the symptoms severity caused by the pathogen. Finally, a drought experiment was established with young plants of the cultivar 'Picual' grown in pots under greenhouse conditions in order to compare the effects caused by water deficit with those caused by the pathogen infection. In both cases, results show that starch hydrolysis occurred, what indirectly evidence the importance of xylem cavitation in the development of the symptoms caused by V. dahliae but in the water stressed plants no vessel occlusion was detected.

Verticillium wilt, caused by Verticillium dahliae Kleb., is presently the most destructive disease of olive, particularly in Andalucia (southern Spain). ‘Picual’ and ‘Arbequina’ are the dominant cultivars being planted in Spain. Both cultivars are highly susceptible to the defoliating pathotype of V. dahliae when artificially inoculated by root-dipping or stem injection. Conversely, ‘Arbequina’ is considered more resistant than ‘Picual’ based on field observations and farmer's experience. In this study, the differential reaction between of cultivars was confirmed by surveys of naturally infested orchards with different inoculum densities of the pathogen. The average percentage of affected olive trees of ‘Picual’ was 60.2%, while only 13.1% of trees of ‘Arbequina’ showed disease symptoms. Overall, the pathogen caused extensive wilting of branches and defoliation on the trees of ‘Picual’, whereas ‘Arbequina’-infected trees showed chlorotic symptoms and slight defoliation. The relationship between inoculum density and disease incidence fit a logarithmic function for both cultivars. The percentage of affected trees of ‘Arbequina’ per year increased linearly (y = 0.3559x, R2 = 0.5652, and P = 0.0195) with the inoculum density in the soil, whereas this relationship was not observed for the ‘Picual’. Planting density had no effect on disease incidence for any of the two cultivars.

Developing verticillium wilt resistant genotypes is currently a major objective in olive breeding. In this study, 6017 genotypes derived from 48 crosses obtained by open pollination and crosses between olive cultivars, wild olive genotypes and other Olea species and Olea europaea subspecies were individually evaluated for verticillium wilt resistance. More than 800 genotypes were identified as resistant to the disease based on the absence of symptoms. High genetic variability and wide segregation in resistance were observed. The inheritance of resistance was studied, and the best parents and crosses to breed resistant genotypes were identified. According to the results, verticillium wilt resistance in olive appears to be a quantitative trait. The results obtained by comparing the level of resistance between different crosses as well as by estimating heritability suggest that it is possible to breed for verticillium wilt resistance in olive.

The use of continuous and natural lighting was studied to assess the resistance of 12 olive cultivars to the defoliating pathotype of Verticillium dahliae. The plants were inoculated by dipping their bare root system in a conidial suspension. ‘Frantoio’ was used as a moderately resistant control cultivar. Several evaluated cultivars were susceptible to the defoliating pathotype of Verticillium dahliae. However, six of these cultivars were moderately resistant. In most of the evaluated cultivars, continuous lighting allowed for the identification of resistant genotypes during a period that was 3 weeks shorter than that which is normally required in these assessments. This reduction was even greater for the resistant cultivars. The use of continuous lighting could thus be an important tool for use in olive breeding programs, where it is necessary to evaluate many genotypes in short durations and, therefore, to optimize time, space, and labor.

The resistance of 28 Spanish olive cultivars to Verticillium dahliae was evaluated in an experiment conducted under greenhouse conditions, by impregnating plant roots with a semisolid fluid mass of a mixture of culture medium and the conidia and mycelium of the fungus. Five-month-old olive plants were inoculated with a cotton defoliating isolate of V. dahliae. ‘Frantoio’ and ‘Picual’ were used as resistant and susceptible reference cultivars, respectively. Cultivars were assessed on the basis of final values of the area under the disease progress curve, mean severity of symptoms, and mortality at 26 weeks following inoculation. Verticillium wilt disease developed more slowly and reached lower values of these parameters than those normally recorded in previous studies conducted in growth chambers, using root-dip inoculation in a conidial suspension of the pathogen. However, most of the evaluated cultivars exhibited susceptible or moderately susceptible reactions to the infections caused by V. dahliae. In particular, a group of eight cultivars, from the same group as ‘Picual’, such as ‘Manzanilla de Abla’, ‘Manzanilla del Centro’ and ‘Negrillo de Iznalloz’, were significantly more susceptible than ‘Frantoio’. Conversely, ‘Escarabajillo’, ‘Menya’ and ‘Sevillana de Abla’ exhibited a high level of resistance to the disease, no dead plants, and vegetative recovery. Field experiments are currently being carried out to confirm the level of resistance assigned to these last genotypes. If confirmed, these genotypes will act as potential resistant genitors for inclusion in current olive breeding programs or for use as resistant rootstocks.

a b s t r a c t Effective inoculation methods to screen for Verticillium wilt resistance are essential for the development of olive cultivars resistant to this devastating disease. Three inoculation methods, pot immmersion, bareroot dipping and stem injection using a conidial suspension of a highly virulent Verticillium dahliae isolate (named V117) were tested in olive seedlings. The root-dipping inoculation performed the best, and its effectiveness was further tested in seedlings aged 40, 80 and 120 days in two different environments (greenhouse and growth chamber). The root-dipping inoculation of the 40-day-old olive seedlings discriminated between resistant and susceptible genotypes. This early screening is less costly and requires less time and space than the standard inoculation and evaluation methods conducted with older plants. Therefore, we propose the root-dipping inoculation of 40-day-old olive seedlings as a reliable, fast and effective method to select genotypes at a young age that are potentially resistant to V. dahliae. The application of this method has allowed for the screening of more than 8000 genotypes before their evaluation under field conditions. © 2013 Elsevier B.V. All rights reserved.

The resistance of 11 olive cultivars to Verticillium dahliae was assessed in two experimental field trials. One-year-old rooted olive cuttings from the World Olive Germplasm Bank (IFAPA research center, Córdoba, Spain) were planted in a heavily infested field in Utrera (Sevilla province) and in a moderately infested field in Andújar (Jaén province) of southern Spain. Plants were assessed for Verticillium wilt resistance during 22 months based on disease severity and tree growth. Severe disease symptoms were observed 6 months after planting in both trials. Twenty months after planting in the heavily infested soil, V. dahliae had killed nearly all of the trees of ‘Bodoquera’, ‘Cornicabra’, ‘Manzanilla de Sevilla’, and ‘Picual’, demonstrating the elevated risk of planting susceptible cultivars in a soil heavily infested with V. dahliae. ‘Arbequina’, ‘Koroneiki’, ‘Sevillenca’, and especially ‘Frantoio’, ‘Empeltre’, and ‘Changlot Real’ showed a high level of disease resistance. However, all of them were affected by the disease. Although the field results confirmed the level of resistance previously obtained for these olive genotypes under controlled conditions, there were some discrepancies. This information will be useful in managing the disease and also in selecting new cultivars for the breeding of Verticillium wilt resistance.

The effect of irrigation frequency (daily [T1], biweekly [T2], and dryland [T3]) on Verticillium wilt of olive was studied in two fields that were naturally infested with Verticillium dahliae in southern Spain and planted to ‘Picual’ olive. Disease onset (average 61 weeks after planting) and disease incidence (average 75.6%) did not differ among irrigation treatments in both fields. Irrigation consistently increased disease development regarding dryland treatment, but this effect varied over time. In experiment I, T1, the relative area under the disease progress curve was greater on all recording dates (ranging from 15.8 to 33.7) in comparison with T3 (average 6.6). Data for experiment II were similar to this on the most favorable dates for disease (March to April). The T2 treatment value varied over time depending on the season and experimental field, being difficult to differentiate from the values of T1 and T3. Significant correlation between disease incidence and severity increments during spring and fall with the soil water content of the same or previous favorable seasons was observed. Through these correlations, we detected soil water contents of 24.3% (experiment I) and 23.6% (experiment II), where the increments of disease parameters remained at zero. Therefore, scheduling irrigation treatments based on rainfall may be a feasible method for maintaining the soil moisture below levels that favor for disease development., This research was partially funded by Project AGL2007-65766 (Spanish Ministerio de Ciencia e Innovación, co-financed by FEDER of the EU) and by a contract between the University of Córdoba and The Spanish Interprofessional Olive Oil Association (UG 400544). We thank the Oficina Comarcal Agraria “Sierra Morena/Campiña Jaén” (Andújar, Jaén), the Comunidad de Regantes “Ntra. Sra. de los Dolores” (Arjona, Jaén), and R. Jiménez del Río for technical assistance at the plot of experiment I; the Oficina Comarcal Agraria “Las Marismas” (Lebrija, Sevilla) and the Sociedad Cooperativa Andaluza “Agroquivir” (Trajano, Sevilla) for permitting us to collect from their property the infested soil necessary for experiment II; F. Gonzalez for his constant valuable support at both experimental plots; and J. M. Moral, who holds a Talent Hub fellowship launched by the Andalusian Knowledge Agency and cofounded by the European Union’s 7th FP.

From autumn 2012 to autumn 2013 a survey comprising 70 olive orchards was carried out in an Irrigation Community of the central Guadalquivir Valley, (Andalucía, Southern Spain) with the objective of studying the influence of the irrigation dose on Verticillium wilt (Verticillium dahliae Kleb.) of olive (VWO). The 93.1% of the visited orchards were planted with susceptible cultivars to V. dahliae (‘Picual’ and ‘Hojiblanca’). Mean disease incidence (DI) of the orchards accounted for 14.9%. In almost all investigated plantations disease onset occurred after farmers had introduced irrigation. In the case that it was present, the introduction of the irrigation always encouraged VWO development, and DI was positively associated with the number of years of irrigation, showing a linear trend (P = 0.006). VWO was significantly more severe in olive plantations irrigated with high irrigation doses. The 47.1% of olive plantations were irrigated with mean values of 2059.9 (in 2012) and 1793.4 (in 2013) m3/ha, and showed the highest DI (21.42%). Plantations watered with lesser doses (the 28.6%) with average values of 712.5 (in 2012) and 741.5 (in 2013) m3/ha showed a DI significantly lower, which accounted for 13.3%. Finally, dry-land olive orchards (24.3%) showed the lowest DI (4.0%)., Research founded by a contract between the and The Spanish Interprofessional Olive Oil Association (UG 400544).

Verticillium wilt of olive (Olea europaea), caused by the fungus Verticillium dahlia, is the most serious disease in the Mediterranean countries. The use of resistant cultivars or rootstocks is one of the most efficient measures for the control of this disease. Olive shows a wide range of genetic variability useful for finding resistance to the disease. The first step to achieve this objective is the identification of resistance-transmitting genitors, in order to screen many genotypes for resistance to V. dahlia. More than 100 olive seedlings from one crossing and open pollination of different genitors were evaluated for resistance to V. dahlia under controlled conditions. Two-month-old seedlings were inoculated by dipping their root system in a conidial suspension of the highly virulent isolate V117. Disease symptoms (defoliation, wilt, chlorosis and necrosis) were assessed by using a 0-4 scale. Results demonstrated that young olive seedlings can be successfully infected by V. dahlia, showing consistent Verticillium wilt symptoms. Results have provided preliminary information about inheritance of resistance, and they have highlighted ‘Frantoio’ as a suitable genitor to breed for Verticillium wilt resistance in olive. Several olive genotypes were selected for resistance to Verticillium wilt and they will be tested in field conditions.

An experiment was conducted in microplots which were artificially infested with a defoliating isolate of Verticillium dahliae using seven different treatments of inoculum densities ranging from 0 to 10 microsclerotia per gram of soil (ppg). The experiment was conducted in Andalucía (southern Spain), and the susceptible Spanish olive cv. Picual was used to determine the relationship between pathogen inoculum density and the progress of Verticillium wilt of olive (VWO). The inoculum, produced on a sodium pectate cellophane medium, was found to efficiently infect olive trees. Symptoms first appeared 30 weeks after the trees were transplanted into infested soil. Periods of increasing disease incidence in the following seasons and years were mainly during spring and autumn, particularly in the second year after planting. Olive trees exhibited a high susceptibility to the defoliating pathotype of the pathogen, even at very low inoculum levels; in fact, diseased plants were encountered throughout the experiment regardless of the inoculum density treatment. Inoculum densities greater than 3 ppg in the soil resulted in final disease incidence greater than 50% for the trees after 2.5 years. Therefore, these inoculum densities must be considered very high for olive trees. There were no differences in final disease incidence, mean symptom severity, or area under the disease progress curve between plots infested with 10 or 3.33 ppg, whereas other treatments exhibited lower values for each of these disease parameters. The temporal variations of disease incidence and severity were highly correlated for the higher inoculum density treatments, with r2 values ranging from 0.92 to 0.84 for disease incidence and from 0.93 to 0.88 for severity. However, r2 was slightly lower for the treatments involving lower inoculum densities of the pathogen in microplots. The slopes of the linear regression curves were statistically different for nearly all the inoculum density treatments. Positive correlation was found between the initial inoculum density and final disease incidence values after the study period that was accurately explained by mathematical models. The results suggest that susceptible olive cultivars should not be planted in soils infested with virulent defoliating pathotypes of V. dahliae. Results also clarify that inoculum density levels obtained from field soil analyses can be used for establishing a risk prediction system with a view to controlling VWO in olive tree plantations.

The resistance of 26 olive (Olea europaea L.) cultivars to the defoliating pathotype of Verticillium dahliae Kleb. was evaluated in experiments under controlled conditions by stem puncture inoculation. The area under disease progress curve with reference to the maximum value potentially reached over the assessment period (AUDPCP), calculated from the symptom severity values (0–4 rating scale), was the main parameter for assessing resistance, and the percentage of dead plants (PDP), the final mean symptom severity (FMS), and the disease recovery were used as additional parameters. Five cultivars were catalogued as extremely susceptible showing values of AUDPCP, PDP, and FMS higher than 70%, 50%, and 3.0, respectively. Six other cultivars exhibited AUDPCP values ranging from 50% to 70% and FMS higher than 3.0 and were classified as susceptible. A group of 11 cultivars, including ‘Koroneiki’, was moderately susceptible with AUDPCP ranging from 31% to 50%. Finally, ‘Frantoio’, ‘Grosal de Albocafer’, ‘Kato Drys’, and ‘Manzanilla Picúa’ showed maximum AUDPCP values of 22%, no dead plants, and slight symptom severity, being classified as resistant. Stem puncture inoculation was an effective tool for identifying resistance in olive cultivars, but a reduction or a delay in the disease symptom expression of plants was observed regarding the symptom progress observed in plants root dip-inoculated in previous works of resistance assessment. These differences were more pronounced in susceptible than in resistant cultivars.

Experiments were conducted to assess the eradicative effectiveness of a number of organic amendments from various botanical species on microsclerotia (MS) of defoliating (D) and non-defoliating (ND) Verticillium dahliae isolates in infested soil. All the substrates studied significantly diminished the viability of V. dahliae MS when applied as amendments to infested soil. The efficacy was found to depend on the particular species, incubation time, pathogen isolate and whether the soil was sterilized. The effect generally increased with increasing incubation time. The ND isolate of the pathogen was more markedly affected than the D isolate. Organic tissue debris from Diplotaxis virgata, Lavandula stoechas and Thymus mastichina in the non-sterilized soil proved highly effective in reducing the viability of MS of both isolates over time. The D. virgata debris provided the most consistent reduction of MS viability among all types of organic matter and no viable inoculum of either isolate was detected after 6 weeks incubation (wai) in sterilized and non-sterilized soil. The L. stoechas amendment killed the ND V. dahliae isolate propagules in non-sterilized soil. Although the viability of D isolate MS was reduced only 58.6% 3 wai, no viable propagules were detected 6 or 9 wai. Similarly, the control rate with L. stoechas was lower in sterilized than in non-sterilized soil for both isolates. No viable MS were detected after 3 weeks incubation in the non-sterilized soil amended with T. mastichina. Plant debris from C. albidus and C. salvifolius had an intermediate effect on MS viability, but affected the viability of MS of both isolates more markedly in sterilized than in non-sterilized soil. The eradicative effect of organic amendments on V. dahliae MS was confirmed by examining the development of disease incidence and severity of symptoms of Verticillium wilt in cotton plants growing in infested, amended soil. All organic materials reduced the disease incidence and severity in soils infested with both isolates. Thus, no diseased cotton plants were detected in soils that were infested with the ND isolate and amended with any of the organic substrates. Similarly, the C. albidus and L. stoechas treatments completely prevented development of the disease in soil infested with the D isolate. The other organic treatments applied to this isolate strongly diminished the final disease incidence levels, which fell below 16.7%.

The influence of irrigation frequency on the onset and development of Verticillium wilt of olive (VWO) was studied. A split-plot design in microplots with naturally infested soil was established for studying four irrigation frequencies for three olive cultivars with differing levels of disease resistance. Final disease incidence (DI) and mortality in ‘Picual’ plants subjected to daily irrigation treatment (T1) reached values of 100 and 63%, respectively. For Picual-T1 samples, the area under the disease progress curve values were significantly different between 15 December 2012 and 15 July 2013 (14.8 to 42.8%) compared with the average results of the other treatments, which were weekly (T2), biweekly (T3), and deficit (T4) (0.4 to 11.5%). No significant differences between the irrigation treatments were observed in ‘Arbequina’, although the DI progressed consistently (60% in all treatments). In ‘Frantoio’, little disease developed. We conclude that a daily irrigation treatment encourages VWO development in susceptible Picual. Therefore, in susceptible cultivars growing in infested soils under daily irrigation regimes, the extension of irrigation frequency may reduce disease incidence. In the context of an integrated control, the use of resistant cultivars seems to be more relevant than the detrimental effects observed in frequent irrigation schedules., This research was funded by a contract between the University of Córdoba and The Spanish Interprofessional Olive Oil Association. (UG 400544).

The resistance of 33 major olive cultivars to Verticillium dahliae was assessed in four experiments conducted under controlled conditions. Nine-month-old nursery olive plants were inoculated with a defoliating isolate (V117) of V. dahliae. Resistance was evaluated by assessing symptom severity using a 0–4 rating scale and estimating the area under disease progress curves with reference to the maximum value potentially reached over the assessment period (AUDPCP). The percentage of plants killed and that of plants that recovered from the disease were used as additional parameters for classifying the cultivars. Twenty-six of the 33 cultivars were found to be susceptible or extremely susceptible to the defoliating V. dahliae isolate. They exhibited values for AUDPCP higher than 45% with mortality higher than 37.5%. A second group of cultivars, which included `Cipresino', `Koroneiki', `Oblonga', and `Sevillenca', were classified as moderately resistant because they showed an important reduction of the final mean severity values and of values for AUDPCP that ranged from 23% to 42%. `Changlot Real', `Empeltre', and `Frantoio' were resistant to the defoliating isolate of V. dahliae. The resistance of `Empeltre' and `Frantoio', although previously reported, was consistently confirmed, because the two cultivars exhibited no dead plants and had very low values for AUDPCP and final disease. This is the first report of resistance of `Changlot Real' to Verticillium wilt based on ability of the plants to recover from infection and the absence of dead plants. These three cultivars will be useful for inclusion in Verticillium wilt-breeding programs.

Natural recovery from wilt disease symptoms was evaluated in young olive trees root dip inoculated with Verticillium dahliae in a growth chamber over a 12 week period and, later on, when the trees were transplanted in a V. dahliae-free soil in a lathhouse during a period of 127 weeks. Recovery in an individual tree was considered when a plant showed symptom remission after having reached a maximum value of symptom severity. Recovery accounted for 53% of 464 trees that showed wilt symptoms during observations in the two environments. The remaining trees died. Recurrent wilt symptoms were not observed in recovered trees, and recovery was usually accompanied by the production of new green tissues. Recovery was clearly higher in trees inoculated with a non-defoliating (ND) isolate (86.4%) of the pathogen than in those inoculated with a defoliating (D) isolate (23.9%). The percentage of recovery and the level of resistance were significantly correlated. Recovery accounted for 92.1% of the cases in resistant and moderately susceptible cultivars, reaching 100% in plants inoculated with the ND isolate (Table 2); meanwhile it was three times lower (30.1% of the plants) in susceptible and extremely susceptible diseased trees. In the lathhouse, periodical tissue isolations for monitoring the progress of infections over a period of 127 weeks in recovered trees, showed that the pathogen could only be isolated from trees 19 weeks after inoculation. Pathogen isolation was significantly higher from susceptible and extremely susceptible cultivars (84.6%) than from resistant and moderately susceptible ones (33.3%). Results showed that if a tree overcomes infection by pathogen from a single inoculation, and it is able to begin a recovery process, it will not express wilt symptoms again in a pathogen-free environment. The pathogen remained inactive or dead over time in recovered trees. Thus, new infections from rootlets would be necessary for new symptom expression. Recovery from Verticillium wilt is an important natural mechanism that occurs in a high percentage of infected olive trees, and can complement the resistance of the cultivar, particularly in conditions of low inoculum densities of low virulence isolates of the pathogen in the soil.

Resistance of 23 important olive cultivars to Verticillium dahliae has been evaluated in four experiments under controlled conditions. Nine-month-old nursery olive plants were inoculated with a cotton non-defoliating (ND) (V4) or a cotton defoliating (D) (V117) isolate of V. dahliae. Resistance was evaluated by assessing symptom severity using a 0–4 rating scale and estimating the area under disease progress curves. The percentage of plants killed and of those which recovered from the disease were used as additional parameters for including a particular cultivar into a defined category. Most of the evaluated cultivars were susceptible, although at different levels, to both isolates of V. dahliae. All cultivars were more susceptible to the D pathotype than to the ND one. A group of 11 cultivars, including several important Spanish cultivars, were susceptible or extremely susceptible to both pathotypes of V. dahliae. A second group showed differences of resistance depending on the pathotype used. They were susceptible or extremely susceptible to the D pathotype but resistant or moderately susceptible to the ND one. Finally, 'Frantoio', 'Oblonga' and 'Empeltre' were moderately susceptible to the D isolate of V. dahliae and resistant to the ND one. The resistance of 'Empeltre' was evident by the plant ability to recover from infection with either isolates. 'Empeltre' is considered to be a valuable cultivar for inclusion in breeding programmes for resistance to Verticillium wilt.

Research was supported by Projects P08-AGR-03635 (Ministry of Agriculture and Fisheries, Government of Andalusia, Spain) and AGL 2011-30137 (Spanish Ministerio de Ciencia e Innovacion, co-financed by FEDER of the EU).

Verticillium wilt of olive, caused by Verticillium dahliae Kleb., is the most severe disease affecting this crop in most olive growing countries. In this study, the presence of viable structures of V. dahliae in dried inflorescences from wilted olive shoots was investigated. The pathogen was found inside peduncles and flowers, by assessing the number of typical star-shaped microsclerotial colonies formed onto the modified sodium polypectate agar medium. Microsclerotia of V. dahliae were observed inside the peduncles under the stereoscopic microscope. The presence of microsclerotia in these easily decomposable olive tissues shows that infected inflorescences can act as a source of inoculum for Verticillium wilt epidemics.

50 pags, 10 figures, 3 tables, This article is published with open access at Springerlink.com, Olive (Olea europaea L.) is one of the first domesticated and cultivated tree species and has historical, social and economical relevance. However, its future as a strategic commodity in Mediterranean agriculture is threatened by diverse biotic (traditional and new/emerging pests and diseases) and abiotic (erosion, climate change) menaces. These problems could also be of relevance for new geographical areas where olive cultivation is not traditional but is increasingly spreading (i.e., South America, Australia, etc). One of the major constraints for olive cultivation is Verticillium wilt, a vascular disease caused by the soil-borne fungus Verticillium dahliae Kleb. In this review we describe how Verticillium wilt of olive (VWO) has become a major problem for olive cultivation during the last two decades. Similar to other vascular diseases, VWO is difficult to manage and single control measure are mostly ineffective. Therefore, an integrated disease management strategy that fits modern sustainable agriculture criteria must be implemented. Multidisciplinary research efforts and advances to understand this pathosystem and to develop appropriate control measures are summarized. The main conclusion is that a holistic approach is the best strategy to effectively control VWO, integrating biological, chemical, physical, and cultural approaches., Grants AGL2007-65766 and AGL2009-07275 (Spanish Ministerio de Ciencia e Innovación, cofinanced by FEDER of the UE), P07-CVI-02624 and P08-AGR-03635 (Junta de Andalucía, Spain), and FEDERINIA RFP2009-00008-C02-01.

Verticillium wilt of olive (Olea europaea L.), caused by Verticillium dahliae, is nowadays the most serious olive disease in Spain. The disease increments are being observed particularly in young olive plantations, favoured by several factors including inadequate cultural practices and crop production intensification, such as irrigation. Thus, three olive orchards affected by Verticillium wilt, with disease incidence ranging 30-50%, were selected to determine if the drip irrigation could favour the increase of pathogen in soil. Pathogen in soil was quantified in wet zones around the drippers and in dry zones out of them. Inoculum density in all experiments was higher in wet than in dry areas. After 4 months of watering, soil pathogen population increased considerably in wet and dry areas but inoculum density remained higher in the wet soil.

Olive (Olea europaea L.) is a traditional tree crop in Andalucia, southern Spain. Over the past several years, new plantations have been established in areas where other crops susceptible to Verticillium dahliae, such as cotton, sunflower, and vegetables, usually have been grown. Surveys performed from 1989 to 1996 showed that Verticillium wilt, caused by V. dahliae, has become the most important soilborne fungal disease affecting young olive trees in this area (3). Manure is sometime used as a soil amendment before and after tree planting to increase organic matter content of soil. In 1996, manure was used to establish an olive plantation in Sevilla Province. The dung came from sheep fed on harvested fields of cotton, sunflower, and sugar beet. The remaining manure was left in a pile in the field. In 1998, 2 years after planting, many olive plants in the field showed Verticillium wilt symptoms. Samples were collected from the soil and the surfaces and core of the manure pile for isolation and quantitative assessment of the pathogen. Wet-sieving and sodium pectate agar selective medium were used (1). V. dahliae was isolated from both the manure and soil. Mean inoculum density in the samples taken from the pile and soil were 8.4 and 7.2 microsclerotia per gram, respectively. Although it has been demonstrated that V. albo-atrum survives the digestive process in animals (2), this is the first report on dispersion of V. dahliae by manure from sheep fed on infested stubble. The use of manure infested with V. dahliae may contribute to pathogen dispersion and to the increase in Verticillium wilt incidence in olive orchards in Andalucia. Because animal manure is a potential means for V. dahliae transmission, it is recommended that before manure is applied tests be performed, when feasible, to make sure it is pathogen-free. References: (1) E. J. Butterfield and J. E DeVay Phytopathology 67:1073, 1977. (2) H. C. Huang et al. Plant Dis. 70:218, 1986. (3) M. E. Sanchez Hernandez et al. Eur. J. Plant Pathol. 104: 347, 1998.