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In the framework of the official monitoring programme of X. fastidiosa in the Apulian buffer zone and pest-free area, sampling and testing were carried out on olive trees, as primary host species of De Donno strain. The spatial and temporal progression of the infection was studied in the canopy of asymptomatic or low-symptomatic trees of tolerant Leccino and susceptible Ogliarola cvs from 2 infected olive groves. A total of 640 samples/cv from 2 canopy levels (high and low) were analyzed in 4 collection times (June, September, December, March) testing 2 matrices (xylem tissue from twig and from mature leaf with petiole) for qualitative and quantitative analyses of the infection (ELISA, DTBIA, qPCR). The infection rate in the high level of the canopy was higher than in the low level in Ogliarola cv, while the difference between the two levels was less evident in Leccino cv. With respect to serological testing, qPCR showed the best results with both matrices in all testing times except in September with leaves. Twig xylem tissue was also the best matrix for serological tests. However, ELISA and DTBIA showed the lowest infection rate in September using both matrices. The trend in infection rate was different between the 2 cvs over the entire testing period. It remained in a range between 20% and 40% in Leccino cv, while it showed an exponential increase in Ogliarola cv where it reached even 100% in the high part of the canopy. Twig xylem tissue was also the best matrix for pathogen quantification in both cvs, more evident in the high level of the canopy. For both cvs, pathogen concentration increased in the winter period and decreased in the summer period. The decrease continued until December only in Leccino cv with the twig matrix. Based on these results an improved sampling and testing protocol was proposed for monitoring X. fastidiosa in olive trees.

XylAppEU 2.1.3 will be launched at the end of 2019 as the final version of the Android application XylApp_1.2.4 used for the official monitoring of Xylella fastidiosa in Apulia, Italy. It was developed on the basis of the characteristics and input received from stakeholders in three EU pilot areas (Córdoba, Spain; Crete, Greece; Apulia, Italy) and from other project partners. A video tutorial of the previous version (2.1.2) was prepared and launched on the project website and the application download was available for testing between partners. This version considers the following aspects which will be implemented with additional input from partners after testing: (i) analysis of the scenario in the specific country; (ii) identification of the specific needs and resolution conditions in relation to the different users; (iii) definition of the featured applications, constraints, performance, interfaces and any other features to fulfil users’ needs; (iv) active engagement of end-users for the finalisation and validation of the app. The advanced application XylAppEU_2.1.3 was equipped with grids of different sizes (e.g. 100 m x 100 m, 1 km x 1 km) of the three pilot sites, based on the free formats available from the European Environment Agency. With regard to the hardware and other minor functions, different parameters (sensor configuration, connectivity and size of the display) were implemented for the most popular platforms (e.g. Google Android). The following specific features were also implemented: transmission protocols for data sending over the internet; inclusion of grid visualisation of the pilot sites for sample localisation at EU level (ETRS89-LAEA Europe); development of a GIS model for management of EU cartographic data. A new graphic interface has been made, user friendly, which guarantees better functionality and integration of modules, beside guidelines or better management by users. This version allows the acquisition, storage and transmission of data in different formats. As for the previous version, a video tutorial on XylAppEU_2.1.3 will be prepared for dissemination purposes. &nbsp

Citrus variegation ilarvirus (CVV) is one of the oldest viruses reported in Citrus, which produces crinkling, puckering and variegation of leaves in field trees and, occasionally, variegated and deformed fruits. This virus is primarily transmitted by grafting. However, no consistent data are available concerning CVV seed transmission, which is apparently low. The elimination of CVV in citrus genotypes is carried out by shoot-tip-grafting. In the case of seed sources, virus elimination directly in the seeds becomes essential. In cryotherapy, plant pathogens such as viruses, phytoplasmas and bacteria are eradicated by exposing explants to liquid nitrogen. It allows treatment of a high number of samples and results in a high frequency of pathogen-free regenerants. In this work, three polyembryonic Citrus genotypes (sour orange, mandarin and lemon), which were found infected by CVV, were used for trials of virus seed transmission and CVV elimination by dehydration/cryopreservation of the seeds. The dehydrated seeds were placed in cryovials which were then plunged into liquid nitrogen at -196°C. ISSR markers were used to discriminate between zygotic and nucellar plants after treatment. TAS-ELISA and RT-PCR were applied for CVV detection in the seed components before cryopreservation and in the produced seedlings after treatment. CVV seed transmission was assessed for the first time in sour orange and lemon genotypes by serological and molecular means. All tested Citrus genotypes showed CVV infection in both components of the seed plunged in liquid nitrogen. However, all plantlets developed from cryopreserved seeds were CVV-free 2 years after transplanting.

Stigma/style somatic embryogenesis is one of the efficient methods in plant regeneration of most Citrus ssp., without inducing somaclonal variations. Furthermore, somatic embryogenesis from style/stigma proved to be effective in the elimination of the main citrus virus and virus-like diseases. This technique was applied on Algerian citrus collection. Different Citrus species [Citrus sinensis (L.) Osbeck, C. limon (L.) Burm, C. reticulata Blanco, C. paradisi Macfad, C. reshni Hort. ex Tan., C. jambhiri Lush and C. maxima (Burm.) Merrill] were chosen and tested for the presence of the main virus and virus-like agents. Most of the genotypes showed to be infected, mainly by viroid agents. Closed flowers were collected and in vitro cultured on a Murashige and Skoog (MS) medium supplemented with 6- benzylaminopurine. All explants produced callus about 4 to 9 days after culture initiation, whereas embryogenesis occurred after 38 to 150 days in most of the cultured genotypes. Formed embryos were cultured in a single tube before in vivo acclimatization. After sanitary assays, regenerated plants were shown to be free from the agents detected in the mother trees.Key words: Algeria, citrus germplasm, plant regeneration, sanitation, somatic embryogenesis.

Citrus represents one of the most important fruit crops worldwide, in terms of economic value and human nutrition. Among fruit tree species, Citrus is the most affected by graft-transmissible pathogens, mainly represented by virus and virus-like agents. These pathogens cannot be directly controlled by chemical treatments, thus prevention by the use of 'healthy' propagating material becomes essential. Somatic embryogenesis, which is a recently developed technique for Citrus sanitation, has proved to be highly effective in the complete elimination of several citrus virus and virus-like diseases. Therefore, this technique can successfully replace the shoot-tipgrafting sanitation method for the production of healthy citrus mother trees of numerous genotypes used for propagation. Moreover, as the international demand for Citrus is high and the risk of introducing/spreading harmful infectious agents is considerable, in vitro conservation of plant material regenerated by somatic embryogenesis is also very promising for the safe international exchange of citrus healthy germplasm. The regeneration of somatic embryos in Citrus has been obtained from different explant types. Since many Citrus species are polyembryonic, and apomictic embryos are generated in vivo from the nucellar cells, several protocols for the regeneration of somatic embryos in Citrus have been developed using this tissue. However, this system provides poor results with many important monoembryonic genotypes. Several approaches have been tested to regulate somatic embryogenesis in Citrus including modification of plant growth regulators and other additives of culture media, explant type and pre-treatment of explants. To detect the genetic fidelity of regenerated plants, the flow cytometric analyses and several DNA markers have been used. This chapter will describe the wide range of approaches which are currently used to regulate somatic embryogenesis and sanitary aspects in Citrus including the role of plant growth regulators. Moreover, analysis of genetic stability of regenerants is also discussed.

Stigma/style somatic embryogenesis is one of the efficient methods in plant regeneration of most Citrus ssp., without inducing somaclonal variations. Furthermore, somatic embryogenesis from style/stigma proved to be effective in the elimination of the main citrus virus and virus-like diseases. This technique was applied on Algerian citrus collection. Different Citrus species [Citrus sinensis (L.) Osbeck, C. limon (L.) Burm, C. reticulata Blanco, C. paradisi Macfad, C. reshni Hort. ex Tan., C. jambhiri Lush and C. maxima (Burm.) Merrill] were chosen and tested for the presence of the main virus and virus-like agents. Most of the genotypes showed to be infected, mainly by viroid agents. Closed flowers were collected and in vitro cultured on a Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine. All explants produced callus about 4 to 9 days after culture initiation, whereas embryogenesis occurred after 38 to 150 days in most of the cultured genotypes. Formed embryos were cultured in a single tube before in vivo acclimatization. After sanitary assays, regenerated plants were shown to be free from the agents detected in the mother trees.

The use of healthy propagating material is of utmost importance in the control of CTV, which is the most serious virus affecting citrus trees worldwide. All the sanitation methods available are effective in CTV elimination; shoot-tip-grafting (STG) and somatic embryogenesis from stigma and style in vitro culture (SE) are the preferred ones for their numerous advantages. Both techniques are successful in regenerating CTV-free plants, identical to the mother tree, and they can also be applied in the safe international exchange of citrus healthy germplasm. SE is user friendly and can be extensively applied to all Citrus species whenever suitable explants are available; while STG can be applied in the sanitation of clementines and pomelos, which are not regenerated by SE.

Conservation of citrus genetic resources needs an integrated in situ and e x situ approach. In view of preventing dissemination of diseases, it is necessary to resort to ex situ conservation for maintenance of clean material, whereas reinfection may be avoided by using in vivo and in vitro conservation. Following specific guidelines, the collection field fulfilling specific requirements or the insect-proof screenhouse can ensure in vivo conservation of a healthy gene pool as well as pomological traits. For several species, in vitro culture may play a major role as a conservation strategy or even be the only option available. Despite their several advantages, in vitro techniques still display considerable constraints such as genetic instability and length of tissue storage.

Somatic embryogenesis was used to eliminate Citrus psorosis virus (CPsV) from three citrus species (common mandarin, sweet orange and Dweet tangor), all of which regenerated somatic embryos with different embryogenic potential from stigma and style explants. CPsV was detected by double antibody sandwich-indirect-enzyme-linked immunosorbent assay (DASI-ELISA) in explants and embryogenic callus, but was not detested in any of the. plants obtained from somatic embryos, even 24 months after regeneration. Loss of juvenile characters (disappearance of thorns) was observed in the first year of growth and was retained in plants propagated by grafting from thornless stems. Somatic embryogenesis appears to be a very promising technique for the production of healthy citrus stocks.

In this study, the encapsulation technology based on the calcium alginate coating was applied to some Ficus carica L. genotypes. Uninodal microcuttings (3-4 mm long), excised from in vitro proliferating shoots of three Italian cultivars (‘Bifera’, ‘Palazzo’ and ‘Catalanisca’), were employed. The influence of three different plant growth regulators (PGRs): 6-benzylaminopurine (BAP), meta-topolin (MT) and zeatine (ZEA), added to the artificial endosperm, were evaluated. Particularly, the viability, regrowth and conversion parameters of the synthetic seeds were registered after 60 days from the sowing on a hormone-free medium. Results showed that the cultivars ‘Catalanisca’ and ‘Palazzo’ showed the highest viability average rate (90%), followed by ‘Bifera’ (80%). Encapsulated microcuttings regrowth percentage was highest for ‘Palazzo’ (86.7%), followed by ‘Catalanisca’ (81.7%) and ‘Bifera’ (66.7%), while the conversion rate was comparable for the three cultivars (43.4, 40.0 and 48.3%, respectively). A strong interaction between cultivars and PGRs was observed. Moreover, regarding the effect of the PGRs, BAP and MT provided the best results about regrowth and conversion, even if the cultivar ‘Catalanisca’ presented 100% of regrowth with ZEA. To our knowledge, this is the first time, that encapsulation technology has been applied to Italian cultivars of Ficus carica.

The unwanted introduction of the Red Palm Weevil (RPW) coincides with the spread in Malta of two species of Uropodid mites associated with this weevil. Usually, adult RPW carry phoretic forms of C. almerodai which are attached to the underside of elytra, and U. marginata that prefers exposed surfaces of sternum, pygidium, head and legs. These mites use adult RPW to abandon dead palms and to colonize newly infested host-plants. Their role as plant pests is however negligible. Even the plant pathogen conidia, Curvularia which are carried by the mites, seem unable to germinate in palms under laboratory conditions. Both Centrouropoda almerodai and Uroobovella marginata are established in the Maltese Islands.