Olive (Olea europaea ssp. europaea L.) is the most extensively planted fruit crop in the world, covering more than 10.2 million hectares of land, mainly in the Mediterranean Basin. Almost 25% of the total olive trees are grown in Spain, where more than 45% of the world's olive oil is produced (FAO, 2015). Olive industry (oil and table) is a vital sector of Spanish agro-food system with a total value of 3 billion euros, more than 385,000 agricultural holdings, and over half million farmers (INE, 2015)¹ In fact, the global price of olive oil yearly depends to a large extent to the Spanish production.

Olive oil has numerous beneficial properties, mainly associated with its high content of monounsaturated oleic acid (Espósito et al., 2004) albeit other minor components, such as phenolic compounds (i.e., hydroxytyrosol, oleocanthal, and squalene) have also shown substantial benefits for consumer health (Beauchamp et al., 2005). For this reason, olive oil consumption and, concomitantly the olive-growing area, have notably increased worldwide in recent years. Projects to expand the olive-growing surface affect new areas for this crop, such as different provinces of China, India, Saudi Arabia, or the States of Florida and Hawaii in the USA. In many of these plantation areas, the adaptation of the different olive cultivars and the pests and diseases of this crop are unknown. Fortunately, the technicians and farmers have a broad range of cultivars that can be selected according to their adaptation to the target agro-environment area.

Olive tree was probably domesticated over 6,000 years ago in the Middle East, from which it spread until covering the entire Mediterranean Basin. It is also accepted the existence of other diversification centers across Mediterranean Basin (Besnard et al., 2013; Díez et al., 2015). The first farmers selected the most outstanding individuals in each olive-growing area according to their adaptation to the soil and climate and their agronomic characteristics. These original cultivars were subsequently maintained by vegetative propagation and, in general, have remained confined to small areas (Rallo et al., 2005; Díez et al., 2015). The presence of homonymy (different cultivars with the same name in different zones), synonymy (a given cultivar with various names in the areas that it occupies), and wrong denominations is frequent in this crop (Ganino et al., 2006; Trujillo et al., 2013). Thereafter, the exact number of olive cultivars is unknown but it is likely that this number reaches around 2000 (Bartolini et al., 1998). For this reason, and due to the absence of systematic studies, there is a reduced classification of the olive cultivars according to their susceptibility to many diseases, particularly in the case of anthracnose (Moral and Trapero, 2009).

Olive anthracnose, caused by the fungal complex species Colletotrichum acutatum sensu lato (s. lat.), C. boninense s. lat., and C. gloeosporioides s. lat. is the most destructive disease of olive fruit and is widely distributed in many olive-growing regions of the world (Martín and García-Figueres, 1999; Talhinhas et al., 2005; Cacciola et al., 2012; Moral et al., 2014; Schena et al., 2014). About 13 Colletotrichum species, belonging to these three complex species have been described affecting this crop (Talhinhas et al., 2005; Schena et al., 2014; Chattaoui et al., 2016). In general, several Colletotrichum species coexist in each olive-growing region with one or two dominant species and several secondary (Faedda et al., 2011; Moral et al., 2014). For example, the species C. godetiae (syn. C. clavatum) and C. acutatum sensu stricto (s. str.) are dominant in olive orchards of southern Italy (Schena et al., 2017); while C. acutatum s. str. and C. gloeosporioides s. s. are dominant and secondary species, respectively, in Tunisia (Chattaoui et al., 2016).

Olive anthracnose is also called soapy fruit due to its characteristic fruit-rot syndrome with a profuse production of spores in a gelatinous matrix under wet conditions (Moral et al., 2009). In addition to the direct losses due to the premature fall of affected fruit, phytotoxins produced by the pathogen in the rotten fruit cause a second syndrome, the dieback of shoots and branches (Ballio et al., 1969; Moral et al., 2009). Furthermore, even with low fruit-rot incidence (5%), the olive oil coming from affected orchards shows poor chemical and organoleptic characteristics that restrict or impede its commercialization as extra virgin olive oil (Moral et al., 2014).

Both cultivar susceptibility and weather conditions profoundly influence on the olive anthracnose severity (Moral and Trapero, 2012). For example, in the central provinces of Andalusia, where the susceptible cultivars Hojiblanca and Picudo grow, severe epidemics occur if the weather conditions are conducive during the autumn despite the farmers typically applying a 2–3 copper-based fungicide treatments during this period. In southern Portugal, where autumn-winter is more humid than in central Andalusia, severe epidemics occur in super-high-density (hedgerow system) olive orchards planted with the moderately susceptible cultivar Arbequina (Moral and Trapero, 2012; Moral et al., 2014).

Although satisfactory results controlling olive anthracnose can be obtained using inorganic and organic fungicides, field application is not always effective for different reasons, such as: (i) the number of registered fungicides in post-bloom is very small; (ii) Colletotrichum shows a high tolerance to copper, the basic ingredient of the main fungicides used; (iii) and the optimum period for fungicide application is relatively short (Roca et al., 2007; Cacciola et al., 2012; Moral et al., 2014). Since the unripe fruit are resistant to the pathogen, the use of early harvesting before the fruit reaches full ripening or the selection of late-maturing cultivars are efficient and environmentally friendly control measures (Moral et al., 2008). However, these practices have some agronomical inconveniences: (i) if the fruit is immature (not completely black), it usually shows less oil content than mature fruit; (ii) the immature fruit show a high fruit retention force being difficult its mechanical harvest; and (iii) when the fruit still immature (green) during the winter, it is highly sensitive to frost damage (Rallo et al., 2005). Therefore, the use of resistant olive cultivars to anthracnose is the most effective control method, which does not show the previous inconvenience, and can be combined with other measures, such as biological and chemical methods or cultural practices (Moral et al., 2008; Moral and Trapero, 2009; Preto et al., 2017).

The World Olive Germplasm Bank of Córdoba (WOGBC) from Andalusia region, southern Spain, is a magnificent setting to evaluate the susceptibility of the olive cultivars to aerial diseases, including anthracnose, for several reasons: (i) the WOGBC is currently on of the largest olive germplasm banks with more than 900 accessions and 411 cultivars from 24 countries (A. Belaj, unpublished data); (ii) it is located in an endemic anthracnose area (Moral et al., 2015); and (iii) the whole group of olive trees of WOGBC has been identified using Simple Sequence Repeats (SSR) markers, resolving the discrepancies due to misidentification of the trees (Trujillo et al., 2013). During the last 10 years, we have developed and validated laboratory and field methods to evaluate the susceptibility of olive cultivars to anthracnose (Moral et al., 2008; Moral and Trapero, 2009). These methods have been used to assess the resistance of traditional olive cultivars and new ones, coming from the breeding program of the University of Córdoba (UCO) and the Andalusian Institute for Research and Formation in Agriculture and Fishery (IFAPA) (Moral and Trapero, 2009; Moral et al., 2015). While the disease reaction of some olive cultivars is well-known (Moral and Trapero, 2009; Talhinhas et al., 2015), most of cultivars are still unclassified for their resistance to this pathogen (Moral et al., 2014).

Because the olive cultivars from WOGBC have not been systematically screened for resistance to Colletotrichum, the objectives of this study were the following: (i) to assess the susceptibility of cultivars in the WOGBC to anthracnose caused by Colletotrichum spp.; (ii) to identify cultivars representative of each of the susceptibility categories determined in this study; and (iii) to correlate the anthracnose susceptibility with other phenotypic characteristics of cultivars. The phenotypic evaluation of disease reaction is a limiting factor for the selection of olive cultivars by farmers, technicians, and breeders.

Here, we present the largest evaluation of olive cultivars for their resistance to anthracnose, which is considered the most important fruit disease of this crop (Cacciola et al., 2012; Moral et al., 2014). We evaluated the reaction of 308 well-identified cultivars growing in the WOGBC during three epidemic seasons. Among others, two important reasons for which the WOGBC is an excellent experimental plot to evaluate the resistance to C. acutatum s. lat. are: (i) the plot is located in an endemic area for anthracnose due to the proximity of the biggest river of Andalusia (Guadalquivir river); (ii) the plot has a high number of HS olive cultivars that act as an inoculum source of Colletotrichum (Moral et al., 2015). In a previous evaluation, we presented a limited number of olive cultivars based on a single observation (Moral et al., 2005) and without the correct identification of the trees using molecular markers (Trujillo et al., 2013). In the latter study, Trujillo et al. (2013) clarified numerous problems of homonymy, synonymy, and wrong denominations in the WOGBC. For example, the original cultivars Arauco, Cañivano Blanco, Carrasqueño de Lucena, and Razzola, which were planted in the WOGBC, have been identified as synonymous of the cultivars Azapa, Picholine Marocaine, Picual, and Frantoio, respectively (Trujillo et al., 2013).

Olive fruit infection occurs at all stages of its development, from flower bud emergence to ripening (Moral et al., 2009). These infections occur mainly as a result of water-splashed conidia. Also, the anthracnose disease cycle can be influenced by the activity of olive fruit fly (B. oleae), which may increase the fruit's susceptibility by causing wounds or directly act as a spores carrier (Malacrinò et al., 2015). Fruit ripeness increases fruit susceptibility to anthracnose (Moral et al., 2008), with the unripen (or developing) fruit being very resistant to the pathogen, regardless of cultivar (Moral et al., 2008; Cacciola et al., 2012; Moral and Trapero, 2012). Therefore, cultivar resistance is evaluated by inoculating yellowish-green fruit with a spore suspension of the pathogen (Moral et al., 2008; Talhinhas et al., 2015). In any case, the use of this rating scale under field conditions to evaluate the cultivar reaction to the pathogen provides an important (>20-fold) economic saving respect to the artificial inoculation method (Moral, 2009). However, the correct evaluation of the olive cultivars under field conditions can only be conducted during epidemic seasons (Moral and Trapero, 2009), which are sporadic in Mediterranean conditions (Cacciola et al., 2012; Moral and Trapero, 2012). In our case, there were only three epidemic seasons during a period of 10 years. The epidemic intensity during the studied years (Figure 1) was associated with conducive weather conditions for anthracnose, mainly the annual rainfall. Thereafter, the greatest epidemic was in 1997, a wet year (888.4 mm) in Cordoba, which had been preceded by a very wet 1996 (951.1 mm). Furthermore, the epidemic years 2005 and 2006 were moderately wet with an annual rainfall of 548.8 and 560.7 mm, respectively.

In this study, the phenotypic resistance of the cultivars was very stable, as shown by the fact that the experimental error explained only around 10% of the total variance (η²), while the cultivar genotype explained over a 65% of this. This fact implies a high stability of the cultivar response to the pathogen (resistance/susceptibility) during the epidemic years. The season (which includes its weather conditions) and the interaction cultivar-season had also a similar effect as that of the experimental error. We did not conduct anthracnose evaluations during non-epidemic seasons due to the almost complete absence of the disease during these years, even in the HS cultivars (Moral, 2009). In other words, anthracnose is a highly weather-dependent disease (Moral and Trapero, 2012).

As it was previously described, the cultivar reaction to Colletotrichum is a continuous variable ranging from HR to HS cultivars (Moral et al., 2015). Nevertheless, the susceptibility/resistance of the cultivars is more useful and easily understood by the farmers and agronomist if the cultivars are placed into distinct ordinal classes (Pataky et al., 2011). In this study, as in previous studies of olive diseases (Moral et al., 2005; Trapero and López-Doncel, 2005), we classified the cultivars in five categorical groups using a K-means analysis that minimized the variances. The intervals (the ranging values) of each categorical group can shift slightly depending on the number of evaluated cultivars since K-analysis uses a centroid value (average of the data of each group) for each given categorical group (Jain et al., 1999).

In general, susceptible cultivars (MS, S, and HS, total 215 cultivars) were more prevalent than resistant cultivars (R and HR, total 93 cultivars) regardless of country of origin, except for Italy. For Italian cultivars, resistance to the pathogen (C. godetiae) in the WOGB was prevalent. Since Colletotrichum spp. is endemic in north of Italy, most of these Italian cultivars could be selected by the farmers according their resistance to the pathogen (Barranco et al., 2000; Bartolini and Cerreti, 2017). The studied Italian cultivars also show a high degree of genetic homogeneity, for example, they all belong to the chlorotype group E1.1, except the S cultivar Carolea that belongs to the group E1.2 (Besnard et al., 2013). Likewise, all the Italian cultivars in the WOGBC, which belong to the genetic cluster 2 described by Trujillo et al. (2013), are resistant to anthracnose except “Cipressino.”

In the extremes of the resistance/susceptibility, we found the cultivar Dolce Agogia, which showed a complete resistance to the pathogen (i.e., absence of disease symptoms), and the cultivars Acebuchera, Picudo Blanco de Estepa, and Uovo Piccione, in which the fruit-rot incidence was 100%. In this study, we described most of the evaluated cultivars as susceptible (MS, S, or HS) to the pathogen, including the species Olea ferruginea, which was moderately susceptible. Results of present study agree with previous observations for susceptible cultivars: Ascolana tenera, Barnea, Galega vulgar, Gordal sevillana, Hojiblanca, Manzanilla de Sevilla, Morisca, Ocal, Picudo, Sant Agostino, and Verdial de Badajoz; and for the resistant or moderately resistant cultivars: Bical de Castelo Branco, Coratina, Frantoio, Empeltre, Leccino, Manzanilla cacereña, Mixani, and Picual (Barranco et al., 2000; Rallo et al., 2005; Moral et al., 2015; Talhinhas et al., 2015; Bartolini and Cerreti, 2017). Conversely, the published response to anthracnose of some cultivars does not match with our current results. In our study, four of these cultivars [“Abou-Salt Mohazan,” “Azapa” (syn. “Arauco”), “Blanqueta,” and “Cordovil de Castelo Branco”] were more susceptible and seven of them (“Arbequina,” “Cobrançosa,” “Itrana,” “Moraiolo,” “Morrut,” and “Picholine”) were somewhat less susceptible than their respective previous classification (Bartolini and Cerreti, 2017).

Errors in the classification of olive cultivars according to their resistance to anthracnose have been extensively discussed by us and they are usually associated with: cultivar misidentification, effect of the ripeness time during the evaluation moment, low inoculum pressure, unfavorable environmental conditions for disease development, and confusion with other fruit-rot diseases caused by species of Alternaria, Botryosphaeria, Fusarium, or Neofabraea (Moral et al., 2008, 2014, 2015; Moral and Trapero, 2009). Furthermore, the potential interaction between Colletotrichum species (or isolate) and the olive cultivar needs a special mention. In general, this type of interaction has been described with MS and S cultivars, such as “Galega vulgar,” “Cobrançosa,” or “Hojiblanca.” Fortunately, R or HR cultivars, such as “Blanqueta,” “Picual,” or “Frantoio” show a high degree of resistance to the different isolates (Xaviér, 2009; Talhinhas et al., 2015). Concomitantly, some Colletotrichum species (or isolates) tend to be weakly (e.g., C. acutatum s. str. or C. rhombiforme) or highly virulent (e. g. C. godetiae or C. nymphaeae) against a broad range of olive cultivars (Xaviér, 2009; Schena et al., 2014; Talhinhas et al., 2015).

The histogram of resistance/susceptibility of the olive cultivars according to fruit-rot incidence showed that data fit a normal distribution but with a dominance of the susceptible cultivars. Similarly, a substantial deviation from normal distribution among cultivars for different agronomic characteristics has been described, such as yield, ripening data, and oil content (Caballero et al., 2006; León et al., 2008, 2011). Although our study was not intended to address the resistance mechanisms against the pathogen, it suggests a complex and polygenic control of the resistance to Colletotrichum species. In any case, other types of genetic control, including combinations of minor and major genes, could be involved in the resistance of the olive tree to Colletotrichum species (Geffroy et al., 2000). However, the case of major genes mediating the defense against non-biotrophic pathogens is rare (Poland et al., 2009). In addition, phenolic compounds have an important role in the defense of the fruit against fungal pathogen on different crops (Prusky, 1996), including olive (Moral et al., 2015); in this case, and due to phenolic acids derivate from different metabolic pathways in the plant (Boudet, 2007), there is not a clear relationship between genes and phenolic compound. Fortunately for the olive breeding programs, crosses between resistant cultivars produce a high frequency of resistant descendants (Moral et al., 2015).

Likewise, important differences have been observed according to the severity of branch-dieback among olive cultivars. This second syndrome of olive anthracnose is associated with the production of phytotoxins by the pathogen on rotten fruit (Moral et al., 2009, 2014). In our study, both fruit-rot incidence and branch-dieback severity appear correlated albeit some of the cultivars, which showed high values of fruit-rot incidence, did not show dieback symptoms. These results suggest differences in the resistance mechanisms for both syndromes. In the experimental plot, the percentage of isolation of Colletotrichum in semi-selective medium from symptomatic tissues of the olive trees were relatively low (<3%) during spring-summer and it was associated with the rainfall events. Schena et al. (2017), using a duplex qPCR, have described a high colonization of these vegetative tissues from May to October. In our conditions, we have never observed fruiting bodies (acervuli) of the pathogen on leaves or shoots under field conditions (Moral et al., 2009), although they can be induced in humid chamber after 1 month of incubation (Moral et al., 2014). For this reason, we think that these tissues have a limited role as inoculum sources in comparison with affected fruit in southern Spain (Moral and Trapero, 2012; Moral et al., 2014). In Southern Italy, the pathogen is able to infect directly leaves and shoots beside, these latter tissues can be also colonized by the pathogen through the peduncles of rotten fruit (Martelli, 1960). Furthermore, acervuli of the pathogen have been described on olive leaves in other countries, such as Australia and Italy (Martelli, 1961; Sergeeva et al., 2008). These differences could be due to variation among pathogen populations, weather conditions, or cultivar resistance.

It is worthy of note that the evaluation of the severity of branch-dieback caused by Colletotrichum sp. may mislead the inexperienced evaluator since other pathogens can cause similar symptoms. In the event of doubt, the evaluator should conduct isolation for other candidate pathogens. For example, we diagnosed Verticillium wilt in more than 20 olive trees belonging to different cultivars in the WOGBC (Morello et al., 2016).

Current trends in planting high-density orchards (which are very conducive for olive anthracnose) and reducing the use of copper-based fungicides are contrary to the high-quality oils demanded by consumers (Moral et al., 2012, 2014; Díez et al., 2016). Thereafter, the selection of less susceptible cultivars to anthracnose is essential for new plantations. The information about the resistance to olive anthracnose that is presented in this study is fundamental for farmers, technicians, and breeders.

In this paper, we evaluated the resistance of 308 olive cultivars to C. acutatum s. lat. during three epidemic seasons under field conditions. However, there is a clear predominance (69.7%) of susceptible cultivars (MS, S, and HS), we have also identified 32 cultivars (10.4%) highly resistant to the pathogen. The most notable cultivar was “Dolce Agogia,” which did not show any anthracnose symptom during the three seasons. This work constitutes the largest evaluation of olive cultivars according to their resistant to C. acutatum to date, albeit the response of other many cultivars to the pathogen is not well-known and, thus, it should be evaluated. For future work, the methodology described in this paper should be also used to evaluate the cultivar response to other aerial fungal pathogen affecting leaves and fruit, such as V. oleaginea and Pseudocercospora cladosporioides, causal agents of peacock spot and cercosporiosis, respectively.

Conceived and designed the study: AT and JM. Performed the evaluation under field conditions: JM, JRV, CX, and LFR. Curator of the WOGBC: JC. Performed the evaluations in controlled conditions: JM. Analyzed the data: JM and AT. Wrote the paper: JM and AT.