*3.1.1. Oleuropein*

96 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

the utilised isolate for inoculation.

until 30 days after wounding (Fig. 3).

The cv. Frantoio and Urano were the most resistant with no symptoms on tested plants, while cvs. Ottobratica and Sant'Agostino were higly susceptible. Further studies are needed for assessing the behaviour of cvs. Arbequina and Arbosana, largely utilised in superintensive olive plantations, that showed a differentiated susceptibility depending on

Vizzarri et al. (2011) also tested a method for evaluating the expression of the genes (*PAL* and *CHS*) involved in defense mechanisms of olive. The level of trascript of these genes showed significant increments after plantlets wounding. Attaining the highest value 9 hours after plantlets wounding. Afterward, the level of transcript of both genes decrease, more evidently for the gene PAL after 24 hours, in any case maintaining high expression levels

**Figure 3.** Time course of the relative transcript level of *PAL* and *CHS* genes in the leaves of stemwounded olive plantlets (cv. Leccino), as compared to unwounded plantlets (from Vizzarri et al., 2011).

cultivars to verticillium wilt could be hypothesised.

The use oh this method permit to verify that the response of plant to the injury is quite rapid, more or less 9-12 hours. Vizzarri et al. (2011) hypothised that the evaluation of the expression level of genes *PAL* e *CHS* for cultivars with different resistance could be important for verifying the role of phenolic methabolism in olive resistance to pathogenes. In fact, recent papers demonstrated that phenolic compounds are very important for modulating resistance/susceptibility of olive cultivars to verticillium wilt. Phenolic response to verticillium wilt is very different in resistant and susceptible cultivars. The resistant cv. Koroneiki showed higher increasing of phenols than susceptible cv. Amfissis when inoculated with verticillium wilt (Markakis et al., 2010). Genes *PAL* e *CHS* play an important role in the biosynthesis of phenolic compounds, then a role in determing resistance of olive Iannotta et al. (2001) investigated the huge olive showing a low susceptibility to olive fly infestations of some cultivars due to the high content of oleuropein within drupes. That cultivars became particularly interesting in respect to cultivars having a low oleuropein content within drupes also when planted in the same environmental and agronomical conditions. Although a correlation between high oleuropein content and low susceptibility of olive cultivars to olive fly infestations is generally accepted, it is nopt clear the mechanism of action of this compound. Some authors hypothesised a mechanism of action against eggs and young larvae of olive fly explicated by oleuropein and their methabolites within the tissue of drupes, causing a reduction of the preimaginal population of this pest. Iannotta et al. (2001) evaluated the amount and the localisation of oleuropein within drupes of ten cultivars selected among them known as low-susceptible and high-susceptible ones. Furthermore, absolute oleuropein has been applied directly on the oviposition sting in order to evaluate its efficacy to control egg hatchling and the following larval development. Results confirm the different behaviour of tested cultivars with cvs. Bardhi i Tirana, Carboncella di Pianacce, Gentile di Chieti and Nociara less susceptible than cvs. Carolea, Nocellara del Belice, Giarraffa, Cucco, Picholine and Cassanese (Table 12). Susceptibility of cultivars is correlated to the amount of oleuropein within drupes. Furthermore, the amount of oleuropein is higher where female lays eggs. The higher amount of this compound in the epicarp found for the low susceptible cultivars seems to be related to genetic characteristics of cultivars more than to phisiological response to olive fle attacks, as demonstrated by comparing the distribution of oleuropein in healthy and infested drupes.


Susceptibility of Cultivars to Biotic Stresses 99

content of drupes is not affected by *B. oleae* attacks, as it is not moved to the damage site. No differences in the oleuropein content were observed between non-infested and infested

**Figure 4.** Results of treatment test with oleuropein of oviposition stings (from Iannotta et al., 2001,

In previous studies, it has been established that the drupe oleuropein content is genetically determined since it hasn't been observed a statically significative difference between oleuropein content in non infested and infested drupes by *B. oleae* belonging to the same genotype (Iannotta et al., 2001). Moreover, it has been proved that the differences in oleuropein amounts are correlated to the different behavior of the cultivars in relation to

Oleuropein was first isolated from olive leaves (Panizzi et al., 1960) where it is present in high levels (Le Tutour and Guedon, 1992). In addition, it occurs throughout the tree and in any constituent part of the fruit (Servili et al., 1999). Oleuropein confers resistance to diseases and to insect infestation of the olive tree (Soler-Rivas et al., 2000). The bactericidal and bacteriostatic activities of oleuropein and its degradation products against many pathogenic microorganisms have been investigated (Hirschman, 1972; Federici and Bongi, 1983) and its in vitro activity has been detected in relation to several bacteria, fungi, viruses, and parasitic protozoans (Hirschman, 1972; Walter et al., 1973; Gourama and Bullerman, 1987; Tassou et al., 1991; Tranter et al., 1993; Tassou and Nychas, 1994, 1995). Oleuropein can also interfere with the synthesis of virus amino acids, prevent viral shedding, budding or assembly at the cell membrane, inhibit viral replication and, in the case of retroviruses, neutralize the production of reverse transcriptase and proteases. Oleuropein is also able to stimulate phagocytosis, as a response of the immune system against pathogenic microorganisms (Hirschman, 1972). A strong chemotactile repulsion exerted by oleuropein in the oviposition of olive fly eggs has been described (Soler-Rivas et al., 2000). Small droplets of olive sap exuded just after oviposition prevent other females from ovipositing on

drupes belonging to the same cultivar (Iannotta et al., 2002).

olive fly attacks (Iannotta et al., 2001, 2002, 2006b).

modified).

**Table 12.** Detailed percentages obtained in the different cultivars concerning olive fly infestation (from Iannotta et al., 2001, modified). Reported values are referred to 100 drupes. Letters indicate significant statistical differences (P<0.01; ANOVA test).

The use of oleuropein directly on oviposition stings confirm the role of control agent of this compound. After ten days from the oleuropein application, within treated sample only the 31% of olive were infested, while within the untreated sample the infested olive were the 65%.

Phenolic composition and concentration are related to genetic features of a given olive cultivar. These genetic features can be used as varietal markers and as indicators of fruit maturation (Esti et al., 1998). Furthermore, a correlation between olive fruit size and oleuropein content has been shown. Small-fruit cultivars are characterised by high oleuropein content (Amiot et al. 1986). Phenolic compounds are important for the defence of plants against pathogens and insect infestations (Haukioja et al., 1985; Hudgins et al., 2003). The antimicrobial activity of phenolic compounds is well documented (Bisignano et al. 1999; Rauha et al., 2000; Proestos et al., 2005; Pereira et al., 2006, 2007).

It has been shown that oleuropein and cyanidine contents are inversely related. During olive fruit maturation the oleuropein content decreases rapidly (Limiroli et al., 1995) while flavonoid content as cyanidine increases (Amiot et al., 1989). In detail, olive fruit maturation consists of three phases: the growth, green maturation and black maturation (Amiot et al., 1989). While in the growth phase an accumulation of oleuropein occurs, in the green maturation phase it decreases. The black maturation phase is characterized by the appearance of anthocyanins and by the progressive decrease of oleuropein levels (Amiot et al., 1989). In Iannotta et al. (2006a) the mean content of both phenolic compounds appears genetically determined. Similar results were observed by Iannotta et al. (2007a, 2007b) confirming a different olive genotype behavior which depends on the genetically determined content of phenolic compounds (Esti et al., 1998). Moreover, the oleuropein content of drupes is not affected by *B. oleae* attacks, as it is not moved to the damage site. No differences in the oleuropein content were observed between non-infested and infested drupes belonging to the same cultivar (Iannotta et al., 2002).

98 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

Sterile oviposition stings (%)

Bardhi i Tirana 8,5A 27,5C 29,60cd 32,89 26,48

Pianacce 9,5A 26,0C 60,04b 70,54 49,55 Gentile di Chieti 9,6A 26,4C 38,82bc 37,69 39,96 Nociara 8,9A 25,2BC 91,91a 141,37 42,45 Carolea 22,7CDE 13,8AB 2,25cd 23,90 20,61 Nocellara del Belice 23,1DE 18,6ABC 40,52bc 48,93 32,12 Giarraffa 23,6DE 16,5AB 11,41d 13,05 9,78 Cucco 26,8E 23,5ABC 21,10cd 23,24 18,87 Picholine 24,1E 17,1ABC 18,80cd 17,36 20,25 Cassanese 27,4E 16,9ABC 14,30d 17,24 11,36 **Table 12.** Detailed percentages obtained in the different cultivars concerning olive fly infestation (from Iannotta et al., 2001, modified). Reported values are referred to 100 drupes. Letters indicate significant

The use of oleuropein directly on oviposition stings confirm the role of control agent of this compound. After ten days from the oleuropein application, within treated sample only the 31% of olive were infested, while within the untreated sample the infested olive were the

Phenolic composition and concentration are related to genetic features of a given olive cultivar. These genetic features can be used as varietal markers and as indicators of fruit maturation (Esti et al., 1998). Furthermore, a correlation between olive fruit size and oleuropein content has been shown. Small-fruit cultivars are characterised by high oleuropein content (Amiot et al. 1986). Phenolic compounds are important for the defence of plants against pathogens and insect infestations (Haukioja et al., 1985; Hudgins et al., 2003). The antimicrobial activity of phenolic compounds is well documented (Bisignano et al. 1999;

It has been shown that oleuropein and cyanidine contents are inversely related. During olive fruit maturation the oleuropein content decreases rapidly (Limiroli et al., 1995) while flavonoid content as cyanidine increases (Amiot et al., 1989). In detail, olive fruit maturation consists of three phases: the growth, green maturation and black maturation (Amiot et al., 1989). While in the growth phase an accumulation of oleuropein occurs, in the green maturation phase it decreases. The black maturation phase is characterized by the appearance of anthocyanins and by the progressive decrease of oleuropein levels (Amiot et al., 1989). In Iannotta et al. (2006a) the mean content of both phenolic compounds appears genetically determined. Similar results were observed by Iannotta et al. (2007a, 2007b) confirming a different olive genotype behavior which depends on the genetically determined content of phenolic compounds (Esti et al., 1998). Moreover, the oleuropein

Rauha et al., 2000; Proestos et al., 2005; Pereira et al., 2006, 2007).

Oleuropein (drupe) (mg/g)

Oleuropein (epicarp) (mg/g)

Oleuropeina (mesocarp) (mg/g)

Active infestation (%)

statistical differences (P<0.01; ANOVA test).

Cultivar

65%.

Carboncella di

**Figure 4.** Results of treatment test with oleuropein of oviposition stings (from Iannotta et al., 2001, modified).

In previous studies, it has been established that the drupe oleuropein content is genetically determined since it hasn't been observed a statically significative difference between oleuropein content in non infested and infested drupes by *B. oleae* belonging to the same genotype (Iannotta et al., 2001). Moreover, it has been proved that the differences in oleuropein amounts are correlated to the different behavior of the cultivars in relation to olive fly attacks (Iannotta et al., 2001, 2002, 2006b).

Oleuropein was first isolated from olive leaves (Panizzi et al., 1960) where it is present in high levels (Le Tutour and Guedon, 1992). In addition, it occurs throughout the tree and in any constituent part of the fruit (Servili et al., 1999). Oleuropein confers resistance to diseases and to insect infestation of the olive tree (Soler-Rivas et al., 2000). The bactericidal and bacteriostatic activities of oleuropein and its degradation products against many pathogenic microorganisms have been investigated (Hirschman, 1972; Federici and Bongi, 1983) and its in vitro activity has been detected in relation to several bacteria, fungi, viruses, and parasitic protozoans (Hirschman, 1972; Walter et al., 1973; Gourama and Bullerman, 1987; Tassou et al., 1991; Tranter et al., 1993; Tassou and Nychas, 1994, 1995). Oleuropein can also interfere with the synthesis of virus amino acids, prevent viral shedding, budding or assembly at the cell membrane, inhibit viral replication and, in the case of retroviruses, neutralize the production of reverse transcriptase and proteases. Oleuropein is also able to stimulate phagocytosis, as a response of the immune system against pathogenic microorganisms (Hirschman, 1972). A strong chemotactile repulsion exerted by oleuropein in the oviposition of olive fly eggs has been described (Soler-Rivas et al., 2000). Small droplets of olive sap exuded just after oviposition prevent other females from ovipositing on the same fruit (Girolami et al., 1981; Lo Scalzo et al., 1994). Oleuropein acts by inhibiting the development of olive fly immature stages, especially eggs and first instar larvae during the early ripening period (Iannotta et al., 2002). The higher concentration of oleuropein in the epicarp than in the mesocarp may be due to the biological function of oleuropein in drupe protection against pests (Soler-Rivas et al., 2000). In fact, the epicarp is the interface between the outer environment and the inner olive fruit. Therefore, high levels of oleuropein in the epicarp protect the fruit against olive fly ovideposition (Iannotta et al., 2002).

Susceptibility of Cultivars to Biotic Stresses 101

are characterised by high oleuropein content (Amiot et al., 1986), playing a synergic role in

Interestingly, it has also been observed that many cultivars characterized by low susceptibility to olive fly attacks showed low susceptibility to the fungal pathogen *Spilocaea oleagina* (Cast.) Hugh. and a negative correlation between oleuropein content in olive leaves and fungal infection has been found (Iannotta and Monardo, 2004). In addition, a correlation between *B. oleae* infestation and *Camarosporium dalmaticum* (Thüm.) Zachos & Tzav.-Klon. infection has been established (Iannotta et al., 2007d). Since the same cultivars showed low levels of susceptibility to both parasites, it could be assumed that high levels of oleuropein may play a role also in determining low cultivar susceptibility to fungal disease (Iannotta et

Cyanidine occurs in olive fruits (Servili et al., 1999) and an increase of cyanidine content at the end of the maturation stages of the olive fruit, as a consequence of hydrolytic processes, was found (Vinha et al., 2005). On the reasons of different genotype behavior concerning the susceptibility to olive fly attacks, the direct influence of cyanidine in the drupes could be, in effect, supposed. It is evident in cvs. Cellina di Nardò, Nolca and Termite di Bitetto which register high value of cyanidine, increasing during the season (Iannotta et al., 2006b). When investigated genotypes are cultivated in the same pedoclimatic conditions and samples obtained from them are collected in the same ripening times, it is possible attribute the differences, concerning cyanidine amount, to a strong influence of the different investigated genotypes genetic diversity. It has been observed that the completely pigmented drupes are not very recognizable by *B. oleae* females determining considerable difficulties for their

A role played by cyanidine in resistance to herbivores was additionally assessed (Harborne and Williams, 1998). Significant differences were found among cultivars in relation to active and total infestations and cyanidine content (Iannotta et al., 2006a). Cultivars Ascolana tenera and Nostrana di Brisighella had the highest level of active infestation (34.33% and 32.33%, respectively) while cv. Cellina di Nardò was the least infested (9.83%). In addition, cvs. Frantoio, Gordal sevillana, Koroneiki, Nera di Cantinelle, Nolca, Ogliarola garganica, and Tonda di Strongoli showed low levels of susceptibility to olive fly (lower than 15%). Cultivars Cellina di Nardò, Nolca, and Termite di Bitetto had higher levels of cyanidine than

In a study undertaken in 2005 in an experimental field located on the Ionian coast of Calabria (Southern Italy), Iannotta et al. (2007a) found the lowest susceptibility to olive fly attack for cvs Tonda nera dolce and Bardhi i Tirana (6.67% and 13.50%, respectively). On the contrary, cvs. Carolea, Cassanese, Carboncella di Pianacce, Gentile di Chieti, Giarraffa, Nocellara del Belice, Nociara and Picholine were susceptible with a mean percentage of active infestation ranging from 22.17 to 29.83%. The presence of cyanidine in the first

determining low susceptibility.

al., 2006c, 2007a).

*3.1.2. Cyanidine* 

ovideposition (Caleca, pers. comm.).

other cultivars in the study and had low levels of infestation.

Moreover, the defence response of fruits damaged both by pathogens and mechanical means, is mediated by β-glucosidase; this enzyme hydrolyses the oleuropein, producing highly reactive aldehyde molecules. Olive cultivars with different levels of enzyme activity have differing degrees of susceptibility to the olive fly. This may be related to the ability of the β-glucosidase to produce highly reactive aldehyde molecules in damaged tissues. A strong peroxidase activity is thereafter detected as a consequence of damage (Spadafora et al., 2008). Results obtained by Iannotta et al. (2001) showed that five cultivars (Bardhi i Tirana, Carboncella di Pianacce, Gentile di Chieti, Kokermadh i Berat, and Nociara) with high levels of drupe oleuropein (31.18 – 36.60 g kg-1) had low levels of infestation (lower than 10%). When oleuropein content decreases, a corresponding increase in the amount of damage caused by olive flies occurs. In the same cultivars, Iannotta et al. (2001) found that the percentage of sterile oviposition stings ranged from 25.0 to 27.5%. Similar results were also observed for cultivars Sant'Agostino, Leccino, and partially Frantoio (Basile et al., 2006). Sterile sting numbers and oleuropein content are inversely proportional to infestation (Iannotta et al., 2001). The role of oleuropein in the inhibition of the development of olive fly immature stages has been shown by performing a comparison between untreated olive samples and samples treated with oleuropein belonging to the cv. Carolea. The cultivar Carolea was chosen because it is susceptible to the olive fly. After ten days, infestation levels were 31% and 65%, respectively, in the oleuropein-treated and non-treated samples (Iannotta et al. 2002). The concentration of oleuropein is greater in the epicarp rather than in the mesocarp during the entire ripening process, except in the case of cvs. Gentile di Chieti and Picholine (Iannotta et al., 2002, 2007a). In these varieties, there is a slightly lower content of oleuropein in the epicarp during the early ripening period. In another study no correlation was observed between infestation and oleuropein content (Iannotta et al., 2006a). In fact, olive fly infestation may be different on the same olive cultivar under different environmental conditions (Fontanazza, 2000) inasmuch as the oleuropein content might be affected by climatic trend (Iannotta et al., 2006a). In a study performed in 2005 in an experimental field located on the Ionian coast of Calabria (Southern Italy), it was observed that cv Cellina di Nardò was the least infested by the olive fly in terms of total infestation (17.67%). In contrast, cvs. Ascolana tenera and Nostrana di Brisighella were the most damaged attaining percentages of total infestation at 56.33% and 57.67%, respectively (Iannotta et al., 2006a). This difference is presumably related also to fruit size (Daane and Johnson, 2010). In fact, Cellina di Nardò has relatively small fruits compared to Ascolana tenera and Nostrana di Brisighella. In addition, it has been shown that small-fruit cultivars are characterised by high oleuropein content (Amiot et al., 1986), playing a synergic role in determining low susceptibility.

Interestingly, it has also been observed that many cultivars characterized by low susceptibility to olive fly attacks showed low susceptibility to the fungal pathogen *Spilocaea oleagina* (Cast.) Hugh. and a negative correlation between oleuropein content in olive leaves and fungal infection has been found (Iannotta and Monardo, 2004). In addition, a correlation between *B. oleae* infestation and *Camarosporium dalmaticum* (Thüm.) Zachos & Tzav.-Klon. infection has been established (Iannotta et al., 2007d). Since the same cultivars showed low levels of susceptibility to both parasites, it could be assumed that high levels of oleuropein may play a role also in determining low cultivar susceptibility to fungal disease (Iannotta et al., 2006c, 2007a).
