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

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

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

Arabis mosaic **ArMV X X**  Cherry leafroll **CLRV X X**  Strawberry latent ringspot **SLRSV X X** 

Olive latent 1 **OLV-1 X X** 

Olive leaf yellowing associated **OLYaV X X** 

**PHYTOPLASMAS X X** 

*Verticillium dahliae* **X X** 

*Pseudomonas savastanoi* pv. *savastanoi* **X X** 

*Meloidogyne incognita* **X X**  *Meloidogyne javanica* **X X**  *Pratylenchus vulnus* **X X**  *Xiphinema diversicaudatum* **X X** 

**Table 2.** Pathogens and pests that must be absent in order to obtain the "virus–free" or " virus–tested"

Cucumber mosaic **CMV X** 

Olive latent 2 **OLV-2 X** 

Tobacco necrosis **TNV X** 

sanitary status according to Italian olive certification law (DM 20/11/2006)

**Sanitary status** 

**Virus-tested (VT)** 

**(VF)** 

**Acronym Virus-free** 

*savastanoi*

**VIRUSES:** 

**FUNGI:** 

**BACTERIA:** 

**NEMATODES:** 

X = the absence of this organism must be ascertained

**Harmful organisms** 

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 material.

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 "phytosanitary selection and certification".

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 greenhouses.

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 extraction and SSR marker analysis.

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.

Regenerated explants are subcultured 3 or 4 times every four weeks using the proliferation medium reported by Rugini (1984), prior to transplanting in jiffy pots. For *in vitro* heat therapy, 2 to 3 cm long in vitro plantlets are exposed for 1 to 3 weeks to 38°C. After heat treatment and subcultures, the surviving plantlets are transplanted in jiffy pots.

Phytosanitary Certification 119

**4. Identification of olive pathogens: updates on diagnostic tools** 

distribution of healthy (virus-free or virus-tested) planting material.

of olive-infecting pathogens advances.

future (Christensen et al., 2004; Hodgetts et al., 2009).

Sanitary certification programs require reliable and sensitive diagnostic tests in order to allow for the identification of pathogen-free trees and the assessment of their overall plant production processes. Due to the latency of several infections caused for example by viruses, visual inspections are not reliable and laboratory tests must be performed to certify virusfree or virus-tested materials. Biological tests and serological assays, widely used to detect pathogens affecting other crops like stone fruits, grapes, pome fruits, result ineffective in olive plants due to the absence of differential woody indicators for the bioassays and the low viral titre and/or to the interference by some contaminants. All these factors have made olive tree virus diagnosis very problematic. Luckily, in the last decade several molecular approaches have been developed and improved to detect olive viruses, bacteria and fungi in the propagating materials. Different molecular techniques such as RT-PCR in single/double step or nested, PCR, real time PCR, dot blot hybridization and dsRNA analysis, have been implemented in recent years and drastically improved sensitivity and specificity of oliveinfecting pathogens' diagnosis. Recently, molecular technology has been successfully applied for routine and large scale detection and could easily be transferred to those Countries that intend to develop their olive crops through production, maintenance and

Concerning viruses, RT-PCR assay has proved to be the most rapid, sensitive and reliable technique for detecting an RNA target in infected plants, and in recent years, different protocols have been developed for olive viruses detection (Grieco et al., 2000; Bertolini et al., 2001a, 2003; Pantaleo et al., 2001; Faggioli et al., 2002, 2005). Recently, a one step RT-PCR protocol has been set up and validated in an inter-laboratory ring test (Loconsole et al., 2010) for the diagnosis of the eight most important olive viruses. This should be a starting point for anyone wishing to approach the sanitary selection of olive plants. New and improved diagnostic techniques (e.g. Real Time RT-PCR, multiplex RT-PCR, polyprobe for molecular hybridization) will be continuously developed as the knowledge on the genetics and biology

Phytoplasma detection is now accomplished through nested-PCR on total DNA extracted from olive plants using the protocol of Barba et al. (1998). Gene amplification is performed using a direct PCR with primers P1/P7 (Deng & Hiruki, 1991; Schneider et al., 1995), followed by a nested-PCR with primers R16F2/R2 (Lee et al., 1993). The use of this analysis allows to determine whether plants are affected by phytoplasma, but does not give information about the identity of the pathogen. Identification of phytoplasma can be achieved through restriction fragment length polymorphism (RFLP) analysis, but it is not required for sanitary certification since the certified olive material must be free from all phytoplasmas. In recent years, the diagnostic technique has also been developed and improved for phytoplasmas. Real time PCR protocols for the identification and group characterization of phytoplasmas are now available. Whilst not yet applied to the diagnosis of olive phytoplasmas, these techniques have all the necessary features for this purpose, and there is therefore potential for their use in the near

For shoot tip culture, apexes are excised from well-established *in vitro* cultures maintained on OM or MSM media. Regenerated apexes are subcultered on the same media prior to being transplanted in jiffy pots.

Olive seedlings for micrografting are recovered from seeds soaked for a few seconds in 70% alcohol before the endocarp removal. Kernels are then soaked for 10 min in 0.05% mercuric hydrochloride solution, rinsed three times in sterile water, and placed on wet sterilized paper in petri dishes at 25° in the dark. After 2-3 months, the regenerated seedlings are cut, leaving about 1.0 cm of the epicotyl. Shoot apexes, excised from *in vitro* grown plantlets, are then grafted on the top of decapitated seedlings. After grafting, plants are cultured in rooting medium (Rugini, 1984). Generally less than 70% of the grafts are successful, and only 10% of the plants survive after transplantation in the soil.

Although phytoplasma diseases may be cured by treatment with certain classes of antibiotics and by heat water therapy, such approaches have not been applied to olive plants in order to obtain sanitation from these pathogens. This is because olive trees affected by phytoplasmas are very rare, and during a phytosanitary selection, phytoplasma-free plants can be easily found. Since their transmission by leaf-hopper vectors is ascertained for other crops, growing nuclear stock plants in insect-proof greenhouses ensures also freedom of infection from phytoplasmas.

The detection of *V. dahliae-*free plants must be carried out with great care. A visual diagnosis is in fact insufficient in guaranteeing the absence of this fungus and have recommended the use of new and sensitive diagnostic tools that are now available (see paragraph 4).

In order to prevent infections by *P. savastanoi* pv. *savastanoi* selected materials must be free from symptoms of the disease, and before propagation material is harvested, mother plants must be sprayed with a copper-based treatment to reduce risk of infections by the epiphytic bacterial population.

The candidate nuclear-stock material obtained through the field selection and/or sanitation treatments describe above can enter the certification program upon official approval (see paragraph 5), and genetically and sanitary certified propagative material will be available to growers.

Demand for olive products is constantly increasing in local and foreign markets, stimulating the expansion of olive crops and encouraging the exchange of olive germplasm at an international level. The activation of a selection and certification program is thus crucial to guarantee the quality of the propagative material and reduce risks for pathogen dissemination.
