**2. Systemic pathogens transmissible with the propagative material**

To date, 8 virus-like diseases have been described, and fifteen different viruses (Tab. 1) and five phytoplasmas have been identified in olive plants. The actual Italian olive certification law (DM 20/11/2006) imposes the absence, in the propagation material, of some of the abovementioned pathogens as well as the most dangerous fungus and the most widespread bacterium in olive crops (Tab. 2). An appraisal of other ways of transmission than vegetative propagation if known, the susceptible hosts, effects and diseases caused by these pathogens and Countries where they have been detected in olive is reported below.

*Arabis mosaic virus (ArMV)* is a member of the genus *Nepovirus*, family *Secoviridae* (Sanfaçon et al., 2011). It is transmitted by the longidorid nematode *Xiphinema diversicaudatum,* but there is no evidence of its transmission to olive plants by this vector. The main hosts of this virus are strawberry, hop, *Vitis* spp., raspberry (*Rubus idaeus*), *Rheum* spp., *Sambucus nigra*, sugarbeet, celery, gladiolus, horseradish and lettuce. The most common symptoms induced by ArMV are leaf mottling and flecking, stunting and several forms of deformation including enations. Because of the serious damages caused on some crops this virus is inserted among the "harmful organisms known to occur in the community and relevant for the entire community" in Directive 2000/29/EC and its absence must be determined on plant material of *Fragaria* and *Rubus.* The symptoms vary depending on the host plant but also on the virus isolate, cultivar, season and year. Many plant species infected with ArMV, including olive trees, do not show any symptoms (Martelli et al., 2002). The virus has been reported in olive trees from Italy (Savino et al., 1979), Portugal (Martelli, 2011) Egypt, USA (Saponari & Savino, 2003), Turkey (Çağlayan et al., 2004), Syria (Alabdullah et al., 2005) and Lebanon (Fadel et al., 2005).

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

period of sampling and protocol for testing.

sanitary) material. The certification scheme in general, and phytosanitary in particular, can

Each step of the propagation (descendent filiation) (Pre-basic, Basic and Certified material) must comply with the requirements that are intended to produce and maintain the selected material in the best growing conditions as specified by the enforced phytosanitary regulations. In particular, the sanitary status must be assessed following the officially recognized technical procedures, regarding the list of the target pathogens, type of sample,

Phytosanitary selection requires the use of appropriate diagnostics protocols for pathogen detection. The difficulty in recognising and/or diagnosing virus-infected olive trees during field surveys imposes the use of laboratory tests in order to assess the absence of the target pathogens. Due to the lack of indicators for the biological assays and the unreliability of the ELISA test in olive, the application of molecular diagnostic techniques for viruses, fungi and bacteria detection became, in the recent past, critical for the assessment of the sanitary status of a given selected ecotype. These sensitive and reliable methods are absolutely

The increasing international demand for olive products, and therefore the expansion of olive crops, is stimulating the exchange of olive germplasm in new areas of the world, prompting for the adoption at European and International level of harmonised Certification Programs that reduce risks of pathogen dissemination and ensure the commercialisation of high quality propagative material and, consequently, guarantee high quality olive productions.

To date, 8 virus-like diseases have been described, and fifteen different viruses (Tab. 1) and five phytoplasmas have been identified in olive plants. The actual Italian olive certification law (DM 20/11/2006) imposes the absence, in the propagation material, of some of the abovementioned pathogens as well as the most dangerous fungus and the most widespread bacterium in olive crops (Tab. 2). An appraisal of other ways of transmission than vegetative propagation if known, the susceptible hosts, effects and diseases caused by these pathogens

*Arabis mosaic virus (ArMV)* is a member of the genus *Nepovirus*, family *Secoviridae* (Sanfaçon et al., 2011). It is transmitted by the longidorid nematode *Xiphinema diversicaudatum,* but there is no evidence of its transmission to olive plants by this vector. The main hosts of this virus are strawberry, hop, *Vitis* spp., raspberry (*Rubus idaeus*), *Rheum* spp., *Sambucus nigra*, sugarbeet, celery, gladiolus, horseradish and lettuce. The most common symptoms induced by ArMV are leaf mottling and flecking, stunting and several forms of deformation including enations. Because of the serious damages caused on some crops this virus is inserted among the "harmful organisms known to occur in the community and relevant for the entire community" in Directive 2000/29/EC and its absence must be determined on plant material of *Fragaria* and *Rubus.* The symptoms vary depending on the host plant but also on

be adopted either for worldwide spread varieties or for those locally distributed.

necessary as they are at the basis of efficient and valid certification programs.

**2. Systemic pathogens transmissible with the propagative material** 

and Countries where they have been detected in olive is reported below.

*Cherry leaf roll virus (CLRV)* belongs to the family *Secoviridae*, genus *Nepovirus,* subgroup c. Even if it is classified as a *Nepovirus*, its transmission by nematodes has not yet been demonstrated to date, whereas it effectively occurs by pollen and, in some hosts, very efficiently by seed too. In olive plants, its transmission by means of pollen has not been demonstrated, but has been ascertained by seeds at the rate of 41% (Saponari et al., 2002). CLRV infects many herbaceous, shrubs and woody plants of genera: *Betula, Celtis, Cornus, Fagus, Juglans, Ligustrum, Olea, Populus, Ulmus, Rubus, Sambucus* and *Rheum*. The virus often induces symptoms in ash, birch, cherry, elderberry and walnut including delayed leaf development, chlorotic leaf streaks or spots, as well as dieback of branches or whole trees but it is symptomless in olive trees (Savino & Gallitelli, 1981). Its presence in olive trees was reported in Italy, Portugal, Spain (Martelli, 1999), then in Egypt, USA (Saponari & Savino, 2003), Turkey (Çağlayan et al., 2004), Syria (Alabdullah et al., 2005), Lebanon (Fadel et al., 2005) and recently in Croatia (Luigi et al., 2011), where it has been shown to have a negative impact on olive fruit and virgin oil quality (Godena et al., 2012).

*Strawberry latent ringspot virus (SLRSV)* is an unassigned species in the *Secoviridae* family. It is transmitted by the nematode *X. diversicaudatum* and by seed in several species (Cooper, 1986), but in olive plants, these kinds of means of transmission have not been demonstrated. SLRSV infects strawberry and raspberry, mostly without symptoms but resulting in various degrees of mottle and decline in some cultivars. The virus was isolated for the first time from olive in cv. 'Corregiolo' in Italy (Savino et al., 1979) and later in Portugal (Henriques et al., 1992), Spain (Bertolini et al., 1998), Egypt, USA (Saponari & Savino, 2003), Turkey (Çağlayan et al., 2004), Lebanon (Fadel et al., 2005), Syria (Alabdullah et al., 2005), Croatia (Bjelis et al., 2007), Tunisia (Martelli, 2011) and Albania (Luigi et al., 2009). Small, pearshaped, puckered fruits with deformed kernels (bumpy fruits), narrow and twisted leaves, bushy growth and reduced crop were described in olive trees of cv. 'Ascolana tenera' affected by SLRSV (Marte et al., 1986). Similar symptoms were observed in cvs 'Negrinha' and 'Galega' in Portugal, associated with a severe reduced rooting ability of the cuttings (Henriques et al., 1992). Among 15 different olive cultivars reporting plants being affected by SLRSV in Portugal, only some showed symptoms (Henriques et al., 1992) in agreement with what was observed in Italy (Savino et al., 1979; Marte et al., 1986); no symptoms are apparently associated with SLRSV infections in Spain (Bertolini et al., 1998). Very interesting is the fact that previously, the 'Raggiola' and 'Frantoio' were considered different olive varieties due to morphological and agronomical dissimilarities. A relatively recent study showed that the two cultivars are genetically identical and that their differentiations are due to the constant presence of SLRSV in 'Raggiola' and the repeated SLRSV absence in 'Frantoio' (Fig. 1) (Ferreti et al., 2002). Rooting trials conducted to compare SLRSV-infected 'Raggiola' with virus-free 'Frantoio' showed that the virus does not influence the rooting

rate of olive cuttings (Roschetti et al., 2009), contrary to what had been previously reported in Portugal.

Phytosanitary Certification 111

Jordan (Martelli et al., 1995), Portugal (Felix & Clara, 2002), Egypt, USA (Saponari & Savino, 2003), Lebanon (Fadel et al., 2005), Syria (Alabdullah et al., 2005), Tunisia (Martelli, 2011) and Turkey (Serce et al., 2007). Several OLV-1 isolates have been obtained from symptomless or weakened trees. Since this virus has also been isolated from citrus in Turkey and Italy (Martelli et al., 1996) and from tulips in Japan (Kanematsu et al., 2001) it is reasonable to assume that it, as well as other olive viruses, may also have a larger host

*Olive latent virus 2 (OLV-2)* is the type species of the monotypic genus *Oleavirus*, family *Bromoviridae* (Grieco & Martelli, 1997). OLV-2 was isolated by mechanical inoculation from symptomless olive trees in Apulia, Southern Italy (Savino et al., 1984). It has subsequently been identified in Lebanon (Fadel et al., 2005), Syria (Alabdullah et al., 2005), Croatia (Bjelis et al., 2007) and Tunisia (Martelli, 2011) from symptomless olive cultivars. The host range of OLV-2 was limited to olive trees until castor beans (*Ricinus communis* L.), showing yellowish vein netting and systemic mottling on leaves, were reported in Greece to be infected with

*Olive latent virus 3* (*OLV-3*) is classified as a tentative member of the genus *Marafivirus,*  family *Tymoviridae*. The virus is not mechanically transmitted. Search of possible vectors, *Euphyllura olivina* and *Saissetia oleae,* was not successful even if OLV- 3 was detected by RT-PCR in the psyllid. A survey conducted in the Mediterranean region showed the OLV-3 presence in Italy, Syria, Malta, Tunisia, Portugal, Turkey, Lebanon and Greece with an infection rate average of 30% always in symptomless olive trees *(*Alabdullah et al., 2010).

*Olive latent ringspot virus* (*OLRSV*) is an approved species of the genus *Nepovirus,* family *Secoviridae*. The virus is transmitted by mechanical inoculation and the existence of a natural vector is unknown. OLRSV is latent in olive trees, but it causes some symptoms on diagnostically susceptible hosts, such as apical necrosis on *Chenopodium quinoa* and *C. amaranticolor,* and red-rimmed local lesions and malformation on tip leaves of *Gomphrena globosa.* The virus was isolated from asymptomatic olive trees in Lazio, Central Italy (Savino et al., 1983), in Portugal in 1990, in Syria (Alabdullah et al., 2005) and then in Tunisia

*Olive leaf yellowing associated virus (OLYaV)* is an unassigned species in the family *Closteroviridae.* Various studies have been published (Sabanadzovic et al., 1999; Essakhi et al., 2006; Luigi et al., 2010) and are still in progress to define its taxonomic position. OLYaV presence in psyllid *E. olivina* and unidentified mealybugs of genus *Pseudococcus* gave the indication that transmission by these vectors could be possible (Sabanazdovic et al., 1999). The olive leaf yellowing (OLY) disease was recorded for the first time in Italy on cv. 'Biancolilla' (Savino et al., 1996) and it is characterized by a bright leaf yellow discoloration (Fig. 2). A survey conducted in Italy showed that, in old OLYaV-affected olive trees, leaf yellowing symptom is frequently absent (Albanese et al., 2003). The OLY syndrome, consisting of poor fruit set, bright yellow discoloration of the foliage, mottling, necrosis, extensive defoliation and dieback, has been associated to other viruses such as *Olive vein yellowing associated virus (OVYaV)* (Faggioli & Barba, 1995) and *Olive yellow mottle and decline associated virus (OYMDaV)*

range.

this virus (Grieco et al., 2002).

(Martelli, 2011).

**Figure 1.** Phenotypic expression of olive cultivars 'Frantoio' SLRSV-affected, healthy 'Frantoio' and 'Raggiola' SLRSV-affected. See as morphological aspect of 'Frantoio' SLRSV-affected and 'Raggiola' SLRSV-affected are identical (narrow leaves and small inflorescences)

*Cucumber mosaic virus (CMV)* belongs to the genus *Cucumovirus*, family *Bromoviridae*. It is one of the most dangerous virus affecting vegetable plants (about 800 wild and cultivated plant species are its hosts). CMV induces important vegetative and productive reductions (up to 100% in plants such as tomato and pepper). When this virus affects herbaceous plants, it is transmitted very efficiently by 75 different aphid species and with varying efficiency by seed. CMV infection in olive is symptomless and its transmission by aphid vectors to/from olive has not yet been proven. It was isolated the first time from olive trees by Savino and Gallitelli (1983) in Italy. This report was confirmed in Portugal by Rei et al. (1993) who detected CMV alone, as well as together with SLRSV. CMV in olive trees was also found in Spain (Bertolini et al., 1998), Turkey (Çağlayan et al., 2004), Syria (Alabdullah et al., 2005), Croatia (Bjelis et al., 2007), Tunisia (Martelli, 2011) and recently in California (Al Rwahnih et al., 2011).

*Olive latent virus 1 (OLV-1)* is a member of the genus *Necrovirus*, family *Tombusviridae*. The virus is one of the few viruses detected in olive trees that is transmitted by seed (at a rate of 82%) (Saponari et al., 2002). It was detected in olive trees in Italy (Gallitelli & Savino, 1985), Jordan (Martelli et al., 1995), Portugal (Felix & Clara, 2002), Egypt, USA (Saponari & Savino, 2003), Lebanon (Fadel et al., 2005), Syria (Alabdullah et al., 2005), Tunisia (Martelli, 2011) and Turkey (Serce et al., 2007). Several OLV-1 isolates have been obtained from symptomless or weakened trees. Since this virus has also been isolated from citrus in Turkey and Italy (Martelli et al., 1996) and from tulips in Japan (Kanematsu et al., 2001) it is reasonable to assume that it, as well as other olive viruses, may also have a larger host range.

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

in Portugal.

al., 2011).

rate of olive cuttings (Roschetti et al., 2009), contrary to what had been previously reported

**Figure 1.** Phenotypic expression of olive cultivars 'Frantoio' SLRSV-affected, healthy 'Frantoio' and 'Raggiola' SLRSV-affected. See as morphological aspect of 'Frantoio' SLRSV-affected and 'Raggiola'

*Cucumber mosaic virus (CMV)* belongs to the genus *Cucumovirus*, family *Bromoviridae*. It is one of the most dangerous virus affecting vegetable plants (about 800 wild and cultivated plant species are its hosts). CMV induces important vegetative and productive reductions (up to 100% in plants such as tomato and pepper). When this virus affects herbaceous plants, it is transmitted very efficiently by 75 different aphid species and with varying efficiency by seed. CMV infection in olive is symptomless and its transmission by aphid vectors to/from olive has not yet been proven. It was isolated the first time from olive trees by Savino and Gallitelli (1983) in Italy. This report was confirmed in Portugal by Rei et al. (1993) who detected CMV alone, as well as together with SLRSV. CMV in olive trees was also found in Spain (Bertolini et al., 1998), Turkey (Çağlayan et al., 2004), Syria (Alabdullah et al., 2005), Croatia (Bjelis et al., 2007), Tunisia (Martelli, 2011) and recently in California (Al Rwahnih et

*Olive latent virus 1 (OLV-1)* is a member of the genus *Necrovirus*, family *Tombusviridae*. The virus is one of the few viruses detected in olive trees that is transmitted by seed (at a rate of 82%) (Saponari et al., 2002). It was detected in olive trees in Italy (Gallitelli & Savino, 1985),

SLRSV-affected are identical (narrow leaves and small inflorescences)

*Olive latent virus 2 (OLV-2)* is the type species of the monotypic genus *Oleavirus*, family *Bromoviridae* (Grieco & Martelli, 1997). OLV-2 was isolated by mechanical inoculation from symptomless olive trees in Apulia, Southern Italy (Savino et al., 1984). It has subsequently been identified in Lebanon (Fadel et al., 2005), Syria (Alabdullah et al., 2005), Croatia (Bjelis et al., 2007) and Tunisia (Martelli, 2011) from symptomless olive cultivars. The host range of OLV-2 was limited to olive trees until castor beans (*Ricinus communis* L.), showing yellowish vein netting and systemic mottling on leaves, were reported in Greece to be infected with this virus (Grieco et al., 2002).

*Olive latent virus 3* (*OLV-3*) is classified as a tentative member of the genus *Marafivirus,*  family *Tymoviridae*. The virus is not mechanically transmitted. Search of possible vectors, *Euphyllura olivina* and *Saissetia oleae,* was not successful even if OLV- 3 was detected by RT-PCR in the psyllid. A survey conducted in the Mediterranean region showed the OLV-3 presence in Italy, Syria, Malta, Tunisia, Portugal, Turkey, Lebanon and Greece with an infection rate average of 30% always in symptomless olive trees *(*Alabdullah et al., 2010).

*Olive latent ringspot virus* (*OLRSV*) is an approved species of the genus *Nepovirus,* family *Secoviridae*. The virus is transmitted by mechanical inoculation and the existence of a natural vector is unknown. OLRSV is latent in olive trees, but it causes some symptoms on diagnostically susceptible hosts, such as apical necrosis on *Chenopodium quinoa* and *C. amaranticolor,* and red-rimmed local lesions and malformation on tip leaves of *Gomphrena globosa.* The virus was isolated from asymptomatic olive trees in Lazio, Central Italy (Savino et al., 1983), in Portugal in 1990, in Syria (Alabdullah et al., 2005) and then in Tunisia (Martelli, 2011).

*Olive leaf yellowing associated virus (OLYaV)* is an unassigned species in the family *Closteroviridae.* Various studies have been published (Sabanadzovic et al., 1999; Essakhi et al., 2006; Luigi et al., 2010) and are still in progress to define its taxonomic position. OLYaV presence in psyllid *E. olivina* and unidentified mealybugs of genus *Pseudococcus* gave the indication that transmission by these vectors could be possible (Sabanazdovic et al., 1999). The olive leaf yellowing (OLY) disease was recorded for the first time in Italy on cv. 'Biancolilla' (Savino et al., 1996) and it is characterized by a bright leaf yellow discoloration (Fig. 2). A survey conducted in Italy showed that, in old OLYaV-affected olive trees, leaf yellowing symptom is frequently absent (Albanese et al., 2003). The OLY syndrome, consisting of poor fruit set, bright yellow discoloration of the foliage, mottling, necrosis, extensive defoliation and dieback, has been associated to other viruses such as *Olive vein yellowing associated virus (OVYaV)* (Faggioli & Barba, 1995) and *Olive yellow mottle and decline associated virus (OYMDaV)*

(Savino et al., 1996), but their presence on olive trees was very rare. On the other hand, OLYaV seems to be one of the most widespread olive viruses: in Italy it infects more than 60% of southern Italy olive cultivars (Faggioli et al., 2005) and it has also been reported in high percentages in Israel (Martelli, 2011), Egypt, USA (Saponari & Savino, 2003), Lebanon (Fadel et al., 2005), Spain (Martelli, 2011), Syria (Alabdullah et al., 2005), Albania (Luigi et al., 2009), Croatia (Bjelis et al., 2007), Tunisia (Martelli, 2011) and California (Al Rwahnih *et al.,* 2011). A study on the rooting and grafting capacity of OLYaV-infected 'Carolea' and its respective healthy controls showed that the virus does not influence the rate of rooting of the cuttings and does not interfere with the grafting success rate; positive significant effects in grafting ability were observed on infected material only during a temperature stress, probably due to the reduced water need of infected shoots (Roschetti et al., 2009). Significant difference in vegetative growth was observed between virus-free and OLYaV-infected young olive plants, demonstrating negative OLYaV interference (Cutuli et al., 2011). To date, no other hosts have been found for this virus.

Phytosanitary Certification 113

Lebanon, Syria, Turkey, Tunisia

Albania, Croatia, Egypt, Italy, Israel, Lebanon, Spain, Syria, Tunisia, USA

Croatia, Egypt, Italy, Lebanon, Portugal, Spain, Syria, Tunisia, Turkey,

Croatia, Egypt, Italy, Lebanon, Portugal, Spain, Syria, Tunisia, Turkey,

USA

USA

Turkey, USA

Tunisia, Turkey, USA

Syria, Tunisia, Turkey

*Tobacco mosaic virus (TMV)* belongs to the genus *Tobamovirus,* family *Virgaviridae*. Mechanical transmission to herbaceous indicator plants was possible, but not easily. It was isolated in central Italy from olive trees showing vein banding, discolorations along the main veins, severe defoliation and decline (Triolo et al., 1996). However, there is no conclusive evidence

*Tobacco necrosis virus (TNV).* Viruses with properties similar to those of TNV were first detected in symptomless olive trees by Félix & Clara (2002) in Portugal. One isolate was studied further, revealing its identity as TNV-D species (Cardoso et al., 2004). However, further genomic characterization of this isolate led to its classification as a new species in the *Necrovirus* genus named *Olive mild mosaic virus (OMMV)* (Cardoso et al., 2005). To date, it is not clear if TNV-D can be considered among the viruses isolated from olive trees, even if recent data shows the presence of this species in olive trees (Cardoso and colleagues deposited the complete genome sequence of a TNV-D isolate from olive trees in the Gene Bank, accession number FJ666328). Virions are readily transmitted by mechanical inoculation and naturally by the fungus *Olpidium brassicacae.* TNV has a wide host range that includes monocotyledonous and dicotyledonous plants, which frequently cause

**Acronym Virus species Genus Geographical distribution** 

OLRSV *Olive latent ringspot virus Nepovirus* Italy, Portugal, Syria, Tunisia

OVYaV *Olive vein yelloving associated virus Potexvirus* Italy OYMDaV *Olive yellov mottling and decline associated virus* Unclassified Italy

OSLV *Olive semilatent virus* Unclassified Italy OMMV *Olive mild mosaic virus Necrovirus* Portugal

TMV *Tobacco mosaic virus Tobamovirus* Italy TNV *Tobacco necrosis virus Necrovirus* Portugal

CLRV *Cherry leafroll virus Nepovirus* 

OLV 1 *Olive latent virus 1 Necrovirus* Italy, Jordan, Portugal, Egypt, USA ,

OLV 2 *Olive latent virus 2 Oleavirus* Italy, Syria, Croatia, Lebanon, Tunisia OLV 3 *Olive latent virus 3 Marafivirus* Greece, Italy, Lebanon Malta, Portugal,

> *Closteroviridae,* unassigned species

*Secoviridae,*  unassigned species

ArMV *Arabis mosaic virus Nepovirus* Egypt, Italy, Lebanon, Portugal, Syria,

CMV *Cucumber mosaic virus Cucumovirus* Croatia, Italy, Portugal, Spain, Syria,

**Table 1.** Viruses identified in olive trees and their geographical distribution (Martelli, 2011; Çağlayan et

that TMV is agent of these symptoms.

necrotic lesions on the roots and leaves.

OLYaV *Olive leaf yellowing associated virus* 

SLRSV *Strawberry latent ringspot virus* 

al., 2009)

**Figure 2.** Yellowing symptoms in an olive cultivar 'Carolea' (a) which tested positive for OLYaV and detail of yellow leaves of the same tree (b)

*Olive semilatent virus (OSLV)* is still unclassified. It was transmitted mechanically from olive tree to *Nicotiana benthamiana* (Materazzi *et al.*, 1996). The main symptom observed in Italy on OSLV-affected olive trees was a very mild chlorotic vein clearing of the leaves, but there is not enough evidence of the etiological involvement of this virus in the disease (Martelli, 1999).

*Tobacco mosaic virus (TMV)* belongs to the genus *Tobamovirus,* family *Virgaviridae*. Mechanical transmission to herbaceous indicator plants was possible, but not easily. It was isolated in central Italy from olive trees showing vein banding, discolorations along the main veins, severe defoliation and decline (Triolo et al., 1996). However, there is no conclusive evidence that TMV is agent of these symptoms.

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

been found for this virus.

detail of yellow leaves of the same tree (b)

(Savino et al., 1996), but their presence on olive trees was very rare. On the other hand, OLYaV seems to be one of the most widespread olive viruses: in Italy it infects more than 60% of southern Italy olive cultivars (Faggioli et al., 2005) and it has also been reported in high percentages in Israel (Martelli, 2011), Egypt, USA (Saponari & Savino, 2003), Lebanon (Fadel et al., 2005), Spain (Martelli, 2011), Syria (Alabdullah et al., 2005), Albania (Luigi et al., 2009), Croatia (Bjelis et al., 2007), Tunisia (Martelli, 2011) and California (Al Rwahnih *et al.,* 2011). A study on the rooting and grafting capacity of OLYaV-infected 'Carolea' and its respective healthy controls showed that the virus does not influence the rate of rooting of the cuttings and does not interfere with the grafting success rate; positive significant effects in grafting ability were observed on infected material only during a temperature stress, probably due to the reduced water need of infected shoots (Roschetti et al., 2009). Significant difference in vegetative growth was observed between virus-free and OLYaV-infected young olive plants, demonstrating negative OLYaV interference (Cutuli et al., 2011). To date, no other hosts have

**Figure 2.** Yellowing symptoms in an olive cultivar 'Carolea' (a) which tested positive for OLYaV and

*Olive semilatent virus (OSLV)* is still unclassified. It was transmitted mechanically from olive tree to *Nicotiana benthamiana* (Materazzi *et al.*, 1996). The main symptom observed in Italy on OSLV-affected olive trees was a very mild chlorotic vein clearing of the leaves, but there is not enough evidence of the etiological involvement of this virus in the disease (Martelli, 1999).

*Tobacco necrosis virus (TNV).* Viruses with properties similar to those of TNV were first detected in symptomless olive trees by Félix & Clara (2002) in Portugal. One isolate was studied further, revealing its identity as TNV-D species (Cardoso et al., 2004). However, further genomic characterization of this isolate led to its classification as a new species in the *Necrovirus* genus named *Olive mild mosaic virus (OMMV)* (Cardoso et al., 2005). To date, it is not clear if TNV-D can be considered among the viruses isolated from olive trees, even if recent data shows the presence of this species in olive trees (Cardoso and colleagues deposited the complete genome sequence of a TNV-D isolate from olive trees in the Gene Bank, accession number FJ666328). Virions are readily transmitted by mechanical inoculation and naturally by the fungus *Olpidium brassicacae.* TNV has a wide host range that includes monocotyledonous and dicotyledonous plants, which frequently cause necrotic lesions on the roots and leaves.


**Table 1.** Viruses identified in olive trees and their geographical distribution (Martelli, 2011; Çağlayan et al., 2009)

*Phytoplasmas* constitute a monophyletic clade within the *Mollicutes* class. Their classification has been possible through the use of restriction fragment length polymorphism (RFLP) analysis and sequencing of the conserved 16S rRNA gene (Lee et al., 1998; Semüller et al., 1998). A variable range of symptoms in olive trees such as shoot proliferation, shortening of internodes, witches'-brooms, little leaves (Fig. 3a), leaf rolling and yellowing, leaf bronzing, phyllody, flower abortion, hypertrophied inflorescences (Fig. 3b), fasciation, erect growth, dwarfing, decline and die-back have been frequently associated with the presence of phytoplasma in Spain, Italy and Iran (Ahangara et al., 2006; Bertaccini et al., 2002; Font et al., 1998; Pasquini et al., 2000). Identification of phytoplasmas detected in olive plants showed they were members of the 16S-IB (Aster yellow), 16S-IC (Clover phyllody), 16Sr-III (Peach X disease), 16S-VA (Elm yellow) or 16S-XIIA (Stolbur) groups and subgroups. The failure to detect phytoplasmas in many symptomatic olive trees leads to doubts on whether these type of alterations could be associated with other causes (Barba, 1993; Camele et al., 1999). Nevertheless, phytoplasmas detected in olive plants are agents causing very well known and severe diseases in other hosts. These include aster yellow, clover phyllody, peach X disease, elm yellow and stolbur in solanaceous plants, as well as grapevine yellow (= Bois Noir). Even if their transmission by leaf-hopper vectors has been proven for some of them (among various host plants but not yet in and from olive plants) their presence in olive plants indicates a serious potential threat for other important crops.

Phytosanitary Certification 115

visible on branches when the bark peeled off. If the cross-section of infected branches or trunks is examined, the brown woody coloration may appear as a ring. Although some plants may die quickly, more commonly trees with only a few wilted branches during a growing season become more severely infected the following year. After the first report of Verticillium wilt in Italy, it has later been detected in Algeria, Arizona, California, Egypt, France, Greece, Iran, Malta, Marocco, Syria, Spain and Turkey (Bubici & Cirulli, 2011). Another species, *V. albo-atrum,* may occasionally cause the same disease in olive plants.

**Figure 4.** Yellow leaves, defoliation and wilt of olive caused by *V. dahliae* (photo by Antonio Ippolito)

of its causal agent to colonize the phylloplane of the tree.

The bacterium *Pseudomonas savastanoi* pv*. savastanoi* causes the most frequent disorder occurring in olive plants known as olive knot disease. The disease manifests itself through the growth of tubercles (Fig. 5) , which either appear individually, or in clusters on any part of the plant, but most commonly on twigs, young branches and around wounds on the main trunk. Knots can damage the stem structure and can deform the scaffold of the tree if infection is severe during the early stages of the tree. This may become a serious problem in nurseries that grow olive plantlets for marketing. *P. savastanoi* causes a similar disease in other plants as oleander, ash, jasmine, Japanese privet, *Forsythia* spp., *Phyllirea* spp.*, Retama sphaerocarpa, Rhamnus alathernus* and myrtle (Surico & Marchi, 2011). This bacterial disease is present in all areas of the world where olive plants are cultivated. This is due to the ability

**Figure 3.** Shortened internodes, witches'-brooms and little leaves (a); hypertrophied inflorescences (b) on olive trees affected by phytoplasmas

The fungus *Verticillium dahliae* is a soil-borne pathogen that attacks olive trees (as well as over a hundred woody and herbaceous species), particularly when their roots are stressed. It causes the most severe disease suffered by olive plants, named Verticillium wilt that induces yellow leaves, defoliation (Fig. 4) and death due to the fungus attacking the plants' vascular system. Internally, a dark reddish brown streak on the wood occurs in most plants. This is visible on branches when the bark peeled off. If the cross-section of infected branches or trunks is examined, the brown woody coloration may appear as a ring. Although some plants may die quickly, more commonly trees with only a few wilted branches during a growing season become more severely infected the following year. After the first report of Verticillium wilt in Italy, it has later been detected in Algeria, Arizona, California, Egypt, France, Greece, Iran, Malta, Marocco, Syria, Spain and Turkey (Bubici & Cirulli, 2011). Another species, *V. albo-atrum,* may occasionally cause the same disease in olive plants.

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

plants indicates a serious potential threat for other important crops.

on olive trees affected by phytoplasmas

**Figure 3.** Shortened internodes, witches'-brooms and little leaves (a); hypertrophied inflorescences (b)

The fungus *Verticillium dahliae* is a soil-borne pathogen that attacks olive trees (as well as over a hundred woody and herbaceous species), particularly when their roots are stressed. It causes the most severe disease suffered by olive plants, named Verticillium wilt that induces yellow leaves, defoliation (Fig. 4) and death due to the fungus attacking the plants' vascular system. Internally, a dark reddish brown streak on the wood occurs in most plants. This is

*Phytoplasmas* constitute a monophyletic clade within the *Mollicutes* class. Their classification has been possible through the use of restriction fragment length polymorphism (RFLP) analysis and sequencing of the conserved 16S rRNA gene (Lee et al., 1998; Semüller et al., 1998). A variable range of symptoms in olive trees such as shoot proliferation, shortening of internodes, witches'-brooms, little leaves (Fig. 3a), leaf rolling and yellowing, leaf bronzing, phyllody, flower abortion, hypertrophied inflorescences (Fig. 3b), fasciation, erect growth, dwarfing, decline and die-back have been frequently associated with the presence of phytoplasma in Spain, Italy and Iran (Ahangara et al., 2006; Bertaccini et al., 2002; Font et al., 1998; Pasquini et al., 2000). Identification of phytoplasmas detected in olive plants showed they were members of the 16S-IB (Aster yellow), 16S-IC (Clover phyllody), 16Sr-III (Peach X disease), 16S-VA (Elm yellow) or 16S-XIIA (Stolbur) groups and subgroups. The failure to detect phytoplasmas in many symptomatic olive trees leads to doubts on whether these type of alterations could be associated with other causes (Barba, 1993; Camele et al., 1999). Nevertheless, phytoplasmas detected in olive plants are agents causing very well known and severe diseases in other hosts. These include aster yellow, clover phyllody, peach X disease, elm yellow and stolbur in solanaceous plants, as well as grapevine yellow (= Bois Noir). Even if their transmission by leaf-hopper vectors has been proven for some of them (among various host plants but not yet in and from olive plants) their presence in olive

**Figure 4.** Yellow leaves, defoliation and wilt of olive caused by *V. dahliae* (photo by Antonio Ippolito)

The bacterium *Pseudomonas savastanoi* pv*. savastanoi* causes the most frequent disorder occurring in olive plants known as olive knot disease. The disease manifests itself through the growth of tubercles (Fig. 5) , which either appear individually, or in clusters on any part of the plant, but most commonly on twigs, young branches and around wounds on the main trunk. Knots can damage the stem structure and can deform the scaffold of the tree if infection is severe during the early stages of the tree. This may become a serious problem in nurseries that grow olive plantlets for marketing. *P. savastanoi* causes a similar disease in other plants as oleander, ash, jasmine, Japanese privet, *Forsythia* spp., *Phyllirea* spp.*, Retama sphaerocarpa, Rhamnus alathernus* and myrtle (Surico & Marchi, 2011). This bacterial disease is present in all areas of the world where olive plants are cultivated. This is due to the ability of its causal agent to colonize the phylloplane of the tree.

Phytosanitary Certification 117

**3. Strategies to control invasive olive pathogens and the importance of** 

Pathogens associated with olive propagative material may be systemic (viruses and phytoplasmas and probably *P. savastanoi* pv. *savastanoi*) or associated with the vascular system (*V. dahliae*) and they are unlikely to be eliminated during the vegetative propagation of an infected source. Accordingly, local and long-distance spread of these pathogens through the movement of infected propagative material has caused a highly threatening worldwide distribution of infectious diseases. The symptomless nature of several olive virus infections may also contribute to the inadvertent propagation and distribution of infected

To avoid disease and/or pathogen dissemination through vegetative propagation, possible remedies include mainly preventive strategies based on the use and propagation of "healthy" mother plants. In fact, in order to attain sanitary improvements of any crop, a system of preventive, protective and often of sanitation measures has to be established and implemented, encompassing a complex series of interventions currently referred to as

In the framework of a phytosanitary and clonal improvement program, the main activities include: (i) field surveys for the selection of olive trees with no apparent disease symptoms and fulfilling the pomological traits of the cultivar; (ii) samples collection for laboratory tests, both for pathogen detection and DNA marker analysis; (iii) molecular tests (RT-PCR, dot blot hybridization and dsRNA analysis) for the detection of viruses included in the certification program; (iv) genetic characterisation using SSR markers; (v) sanitation by heat therapy, meristem tip culture and micrografting in case of no healthy trees being detected for one or more cultivars; (vi) propagation of the candidate nuclear stocks under conditions that ensure freedom from re-infections, usually in insect-proof

Field surveys should be carried out in the main olive-growing areas for the specific cultivar undergoing the clonal and sanitary selection program. Usually mature trees are selected (i.e. 25-year-old) based on visual inspection during spring and autumn. Samples for virus testing consist of 10-15 cuttings collected from 1- to 2-year-old twigs or young leaves for DNA

Despite limited information being available on the application and effectiveness of sanitation protocols on olive plants, *in vivo* and *in vitro* heat therapy, *in vitro* shoot tip culture and micrografting have all been applied in attempts to regenerate OLYaV- and CLRV-free material and some successful results have been obtained (Bottalico et al, 2002). For *in vivo* heat therapy, plants can be grown at 38°C for 3 to 12 months. During heat therapy, 2 to 2.5 cm long shoot tips are excised no earlier than three months from the beginning of the treatment. After surface-sterilization in 0,05% mercuric hydrochloride for 10 min, the shoot tips are placed *in vitro* in petri dishes on different media according to the cultivar [OM (Rugini, 1984); MSM media (Leva et al., 1994)] and grown at 24°C with a 16 h photoperiod.

**phytosanitary certification** 

"phytosanitary selection and certification".

extraction and SSR marker analysis.

material.

greenhouses.

**Figure 5.** Olive knots: rough galls and swellings on twigs and branches caused by *P. savastanoi* pv. *savastanoi*


X = the absence of this organism must be ascertained

**Table 2.** Pathogens and pests that must be absent in order to obtain the "virus–free" or " virus–tested" sanitary status according to Italian olive certification law (DM 20/11/2006)
