**2.5. Resistance to** *Verticillium dahliae*

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

and 5th December).

and agronomic conditions (Fig. 2). The investigation has been performed in 2005 by analyzing 10 cultivars, in the experimental olive plantation made up by a cultivar collection consisting in 20-years old plants. Observations were carried out detecting on 200 drupes per cultivar the infection level (%) of *C. dalmaticum* by direct observation of drupes. Samples were collected in three different period during fruit ripening (03rd October, 04th November

**Figure 2.** Location of CRA-OLI experimental field, where several cultivars are planted under the same agronomical and climatic conditions, on the ionian coast of Calabria (Mirto-Crosia, Cosenza, Italy).

**Table 10.** Mean infected drupes concerning *Camarosporium dalmaticum* infection. Letters indicate significant statistical differences (P<0.01; ANOVA test). (From Iannotta et al., 2007a, modified).

Verticillium wilt, caused by the fungus *Verticillium dahliae* Kleb., is a vascular wilt with a very large host range. Verticillium wilt is increasing in several Mediterranean countries, and it is very difficult to reduce its incidence because is not easy to apply in the field chemical compounds such as fosetyl-Al, directly inoculated within vascular system. Very little is known about biochemical and molecular mechanisms of olive resistance to the Verticillium wilt. Among investigated aspects, it seems to be very important the role of phenolic methabolism (Baidez et al., 2007; Markakis et al., 2010). Vizzarri et al. (2011) investigated the susceptibility of cvs. Arbequina, Arbosana, Frantoio, Ottobratica, Sant'Agostino and Urano by inoculating them with different isolates of *Verticillium dahliae*. Plantlets of 18 months and 160 cm high, has been inoculated by applying a small square of agar with sporulating fungal hyphae on wounded wood. After inoculation plantlets were observed for one year in a greenhouse. The severity of the disese was evaluated by utilising 5 classes of the percentage of damaged plant and the intensity of symptoms: 0%, healthy plant; 20%, plant with large clorosis and moderate foliar symptoms; 50%, severe foliar symptoms and desiccation of vegetative apex; 75%, desiccation of more than half of the plant; 100% dead plant, without defoliation (Colella et al., 2008). Furthermore, Vizzarri et al. (2011) defined a molecular protocol for studying the expression of gene involved in defense mechanisms of the olive. They utilised plants of 14 months of the cv. Leccino, some artificially inoculated as described above and some non inoculated as negative control. Results showed that genetic resistance of tested cultivars, inoculated with different Verticillium isolates, is subordinated to the virulence of the inoculated isolate (Table 11).


**Table 11.** Results of the genotypic susceptibility assay. Number of plants showing a given range of symptoms. Cultivars and isolates used (10 replicates for each cultivar). (from Vizzarri et al., 2011).

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 the utilised isolate for inoculation.

Susceptibility of Cultivars to Biotic Stresses 97

**3. Factors affecting cultivar susceptibility** 

of olive cultivars to biotic stresses.

2007a, 2007b).

*3.1.1. Oleuropein* 

**3.1. The role of phenolic compounds** 

of oleuropein in healthy and infested drupes.

Morphological parameters such as dimensions and coulor are known to be important in determining susceptibility of cultivars affecting female choice for laying eggs. Recently, a great effort is done for determining biochemical factors involved in resistance/susceptibility

Differences in the response of olive cultivars to olive fly infestations have been observed (Gümusay et al., 1990; Iannotta et al., 1999, 2006a, 2007a, 2007b; Pereira et al., 2004; Basile et al., 2006; Rizzo and Caleca, 2006; Daane and Johnson, 2010). Two phenolic compounds, oleuropein and cyanidine, were assessed to determine their role in the reduced susceptibility of certain olive cultivars to olive fly (Iannotta et al., 2006a, 2007a, 2007b). In addition, a positive correlation has been observed between the drupe oleuropein content and a low susceptibility of olive cultivars to olive fly damage such that when the drupe oleuropein content is high, the olive fruit is less susceptible to attack (Iannotta et al., 2006a,

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

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 until 30 days after wounding (Fig. 3).

**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).

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 cultivars to verticillium wilt could be hypothesised.
