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

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

only 10% of the plants survive after transplantation in the soil.

being transplanted in jiffy pots.

infection from phytoplasmas.

bacterial population.

growers.

dissemination.

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

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

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

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

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

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

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

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

use of new and sensitive diagnostic tools that are now available (see paragraph 4).

treatment and subcultures, the surviving plantlets are transplanted in jiffy pots.

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

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 of olive-infecting pathogens advances.

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

Diagnosis of *V. dahliae* is preliminarily performed through an accurate search for foliar symptoms and vascular browning. Foliar chlorosis and necrosis could be due to other causes such as root rot diseases, whereas browning on cross section of stems was sometimes not found. Conclusive detection is attempted by isolating the fungus on agar media from olive tissues and possibly using PCR or nested PCR with *V. dahliae* specific primers (Nigro et al., 2002; Mercado-Blanco et al., 2002) or by Real Time Scorpion PCR (Schena et al., 2004).

Phytosanitary Certification 121

To be classified as such, material must comply with the criteria of quality, plant health, testing methods and procedures, propagation systems and varietal aspects and must have been recognised following official inspections. In addition, propagating material or fruit plants may, in most circumstances, only be marketed by accredited suppliers, whose production methods and facilities meet the requirements of the Directive. Propagating material and fruit plants from Countries outside of the European Union (EU) may only be marketed within the Community if they offer the same guarantees as materials produced in the Community complying with Council Directive 92/34/EEC. Each EU Member State adopts its own enforcement and implementation policies using the EU Directive as a guide. In Italy, it is compulsory that the production and marketing of olive propagating material fulfils the requirements established by the Italian Decree amended on 14/04/1997 in compliance with the aforementioned EU Directive. According to this law, assessment of true to type and certification of sanitary status of olive propagating materials are compulsory and plants are certified as CAC. In this kind of certification plants must be free from *Euzophera pinguis*, *Meloidogine* spp., *S. oleae*, *P. savastanoi* pv. *savastanoi*, *V. dahliae* and all known viruses (15 to date). However, several aspects concerning for example the procedures for inspections and controls are not well defined, leading to misinterpretations and heterogeneous application of the law in the different Italian regions by the regional Phytosanitary Services. Besides this compulsory system, which relies on the propagation of olive mother plants identified by the single nurseryman and found free from the target pathogens, a voluntary certification system has been activated since 1987 at the national level by the Ministry of Agriculture. During the last 10 years, the legislation has been revised, and in late 2006 the revision process was completed with the publication of 2 decrees concerning the revised organisation of the system (DM 24/7/2003, DM 4/5/2006) and 4 decrees concerning the updated official technical operations for the certification of

The DM 20/11/06 provides detailed guidance on the production of olive trees and rootstocks. Plant material produced according to this certification scheme is derived from nuclear-stock plants (also identified as Primary source) officially recognised and registered in the database of certified accessions by the Ministry of Agriculture. The material deriving from the first multiplication of the nuclear-stock material enters in the certification process as Pre-basic material. Once the nuclear-stock has been registered, the breeders or Institutions or Research Centres responsible for its production and selection must keep the material under

The main outlines of the Italian certification scheme (Tab. 3 and Fig. 6) are the following:

a. *Registration of nuclear-stock material*: Breeders or researchers that intend to introduce a new accession for a specific variety in the certification system must provide detailed information about the trueness to type and sanitary controls performed by filling the official forms defined in the DM 20/11/06. The evaluation and eligibility of the registration request is carried out by a technical certification committee authorised by

pommes, stone fruit, olive and strawberry (DM 20/11/06).

conditions that minimise recontamination risks.

the Ministry of Agriculture.

Identification of *P. savastanoi* pv. *savastanoi* is very easy when the typical knots are present on plants. Nevertheless, its presence in latent and systemic form has been reported by Penyalver et al., (2006). The presence of *P. savastanoi* pv. *savastanoi* both as epiphytic and entophytic agent makes its control in the certified material absolutely compulsory; moreover, for a more sensitive and reliable diagnosis, molecular techniques are needed. Recently, molecular protocols of nested-PCR have been set up to obtain reliable diagnoses of latent infections (Bertolini et al., 2001b). This is also possible (and has been done) simultaneously with four other olive viruses (Bertolini et al., 2003).
