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[EN] Citrus tristeza virus (CTV), a phloem-restricted closterovirus infecting citrus, encodes three different silencing suppressors (p25, p20 and p23), one of which (p23) is a pathogenicity determinant that induces aberrations resembling CTV symptoms when expressed ectopically in transgenic citrus hosts. In this article, the effect of p23 ectopic expression on virus infection was examined in sweet orange (SwO), a highly susceptible host, and sour orange (SO), which severely restricts CTV cell-to-cell movement. Transgenic plants of both species ectopically expressing p23, or transformed with an empty vector, were graft inoculated with the mild CTV isolate T385 or with CTV-BC1/GFP, a clonal strain derived from the severe isolate T36 carrying the gene for the green fluorescent protein (GFP). CTV distribution in infected tissues was assessed by direct tissue blot immunoassay and fluorescence emission, and virus accumulation was estimated by quantitative real-time reverse transcriptase-polymerase chain reaction. CTV accumulation in p23-expressing and control SwO plants was similar, whereas the viral load in transgenic SO expressing p23 was 10-10(5) times higher than in the cognate control plants. Although few infection foci composed of a single cell were observed in the phloem of CTV-infected control SO, the number of foci in p23-expressing plants was higher and usually comprised two to six cells, indicating viral cell-to-cell movement. CTV was detected in mesophyll protoplasts and cells from infected SO and SwO expressing p23, but not in similar protoplasts and cells from infected control plants. Our results show that the ectopic expression of p23 enables CTV to escape from the phloem and, in addition, facilitates systemic infection of the resistant SO host. This is the first report of a viral-encoded protein that enhances virus accumulation and distribution in woody hosts., We thank J. E. Peris and J. Juarez for excellent technical assistance, Dr E. Marco-Noales for help with confocal microscopy and Dr W. O. Dawson (University of Florida) for providing the GFP-tagged T36 strain CTV-BC1/GFP. CF is a recipient of a post-doctoral Ramon y Cajal contract from the Ministerio de Ciencia e Innovacion (MICINN). This research was supported by grants AGL2009-08052, co-financed by Fondo Europeo de Desarrollo Regional (FEDER) and MICINN, and Prometeo/2008/121 from the Generalitat Valenciana.

The expression of engineered single-chain variable fragments specific to the NIb RNA replicase of Plum pox virus (PPV) (scFv2A) in transgenic plants was successfully used as a strategy to interfere with viral infection. Different scFv2A fusion proteins were constructed to target those subcellular compartments, such as the cytosol, endoplasmic reticulum (ER) membrane structures and the nucleus, where NIb protein presumably accumulates. Several transgenic lines of Nicotiana benthamiana plants expressing the scFv2A targeted to the cytosol (2A lines), ER (6K2 lines) and nucleus (NLS lines) were obtained. The protective effect of scFv expression was determined by mechanical virus inoculation in five 2A, three 6K2 and four NLS transgenic lines. The strongest resistance was afforded with the 2A-3 (six non-infected plants out of 10), 6K2-1 (17 out of 33) and NLS-11 (16 out of 19) transgenic lines. The success of this interference with PPV infection opens new possibilities for the control of this RNA virus and could be exploited not only to confer resistance in transgenic plants, but also to elucidate the role of the non-structural NIb protein in different cell compartments during viral infection.

Plum pox virus (PPV) o virus de la sharka produce importantes daños y pérdidas económicas en frutales de hueso. En este trabajo se propone probar una nueva y prometedora estrategia de resistencia a la enfermedad de la sharka basada en el uso de anticuerpos recombinantes. La expresión constitutiva de genes o fragmentos de genes de anticuerpo (anticuerpos recombinantes o rAbs) específicos de proteínas virales se ha demostrado que puede inmunomodular o interferir con la infección viral (Tavladoraki y col, 1993). La eficacia de esta estrategia depende principalmente de la naturaleza del antígeno y/o epítopo reconocido por el fragmento de anticuerpo recombinante, de la correcta expresión y acumulación del anticuerpo recombinante y del compartimento celular al que vaya dirigido. El objetivo de este trabajo ha sido evaluar el efecto sobre la infección viral de la expresión de anticuerpos recombinantes específicos de proteínas del virus de la sharka en plantas transgénicas de una especie modelo (Nicotiana benthamiana) como paso previo a su uso en Prunus cuando estos puedan ser transformados y regenerados con éxito.

Desde hace 12 años, los laboratorios de Transformación Genética y Cultivo de Tejidos Vegetales del Departamento de Protección Vegetal y Biotecnología del IVIA, estamos trabajando en el desarrollo de la tecnología de transformación genética aplicada a cítricos y en investigar sus posibilidades de utilización para la mejora tanto de patrones como de variedades. Nuestra intención en este artículo es resumir las investigaciones llevadas a cabo y destacar los logros más importantes obtenidos hasta la fecha. En el contexto de la imperiosa necesidad de mejora de nuestros cítricos para poder seguir siendo competitivos en los mercados internacionales, es necesaria la mejora genética y, entre las tecnologías existentes, la transformación genética de plantas ofrece enormes posibilidades, ya que permite introducir caracteres únicos en genotipos de élite sin alterar su fondo genético. Por ejemplo, se podría incorporar a naranjo amargo un gen (o unos pocos genes) de resistencia a tristeza, de manera que obtuviésemos un naranjo amargo resistente a este virus. Además, los caracteres de mejora no tienen que proceder necesariamente de cítricos, sino que pueden ser aislados de otros organismos vivos. Así se podría conseguir resistencia a ciertas plagas utilizando genes procedentes de bacterias.

Citrus are subject to many biotic and abiotic stresses and markets are continuously requiring fruits of higher quality. These pose important problems in most citrus growing areas that can only be solved with the establishment of citrus improvement programs to recover new and healthy genotypes to be used as rootstocks and varieties. Success of traditional citrus breeding strategies is limited by the peculiar genetic and reproductive characteristics of citrus. Biotechnology offers different approaches that can overcome many of these limitations. In this paper the following technologies used in Spain are reviewed: a) Shoot tip grafting in vitro. It is used worldwide to recover plants free of all known citrus pathogens, and already hundreds of millions of trees originated in this technique have been planted in the field; b) Embryo rescue. It is used in large programs to recover triploid seedless hybrid varieties from aborted seeds from 2n x 4n and 2n x 2n crosses; c) Protoplast fusion. It allows the production of allotetraploid hybrids among sexually compatible and incompatible parents to be used as rootstocks or as parentals for interploid breeding. Fusions between diploid and haploid cell lines are also used to produce triploid hybrids. d) Genetic transformation. Efficient protocols for transformation of several genotypes have been developed and introduction in citrus of genes of potential agronomical interest has been accomplished.

Actualmente, todos los nuevos patrones y variedades que se están produciendo en el mundo se someten a procesos de patente y su utilización está muchas veces restringida por intereses comerciales. Además, en diversos países como EE.UU., Japón, Israel, Francia e Italia se están realizando grandes esfuerzos en la aplicación de nuevas biotecnologías para la mejora de los cítricos y las tecnologías desarrolladas también serán objeto de patente.

En los últimos años, se está planteando en los países citrícolas más avanzados la utilización de diversas biotecnologías complementarias a la mejora convencional para tratar de conseguir genotipos más tolerantes o resistentes a patógenos de cítricos. Entre ellas, la transformación genética es especialmente prometedora, ya que permite la incorporación de caracteres específicos en genotipos de élite sin alterar su fondo genético. Nuestro grupo está trabajando en la incorporación de transgenes en cítricos para tratar de conseguir mayor tolerancia al oomiceto Phytophthora sp. y resistencia al virus de la tristeza de los cítricos (CTV).

The p25 coat protein (CP) gene of Citrus tristeza virus (CTV) was incorporated to Mexican lime plants and forty-two transgenic lines were produced, 25 containing the p25 CP gene of the severe CTV strain T-305 and 17 with that of the mild strain T-317. When plants propagated from each transgenic line were graft-inoculated with CTV T-305 or aphidinoculated with T-300, two types of response to viral challenge were observed: some lines developed CTV symptoms similar to those of non-transgenic controls, whereas others exhibited protection against the virus. This protection consisted of a proportion of plants, ranging from 10 to 33%, that were resistant to CTV, and the rest of them that showed a significant delay in virus accumulation and symptom onset. Protection was efficient against non-homologous CTV strains and was generally accompanied by high accumulation of p25 CP in the protected lines, which suggest a CP-mediated protection mechanism in most cases. This is the first report demonstrating pathogen-derived resistance in transgenic plants against a Closterovirus member in its natural host.

The 23 kDa protein (p23) coded by the 3'-terminal gene of Citrus tristeza virus (CTV), a member of the genus Closterovirus with the largest genome among plant RNA viruses, is an RNA-binding protein that contains a motif rich in cysteine and histidine residues in the core of a putative zinc-finger domain. On this basis, a regulatory role for CTV replication or gene expression has been suggested for p23. To explore whether over-expression of this protein in transgenic plants could affect the normal CTV infection process, transgenic Mexican lime plants were generated carrying the p23 transgene, or a truncated version thereof, under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Constitutive expression of p23 induced phenotypic aberrations that resembled symptoms incited by CTV in non-transgenic lime plants, whereas transgenic plants expressing the p23 truncated version were normal. The onset of CTV-like symptoms in p23 transgenic plants was associated with the expression of p23, and its accumulation level paralleled the intensity of the symptoms. This demonstrates that p23 is involved in symptom development and that it most likely plays a key role in CTV pathogenesis. This is the first case in which a protein encoded by a woody plant-infecting RNA virus has been identified as being directly involved in pathogenesis in its natural host. This finding also delimits a small region of the large CTV genome for the future mapping of specific pathogenic determinants.

Very active programs to produce new mandarin varieties are being carried out in several countries using different approaches and technologies (diploid and triploid breeding, irradiation, somatic hybridization, genetic transformation, etc.). The objective of this workshop was to discuss about the most important breeding methods, the main limitations of the different approaches and methodologies, which is the challenge of new mandarin varieties in the next future and also their relationship with the legislation related to essentially derived varieties that in some cases may preclude the protection of breeder’s rights of new varieties.

Spain is the largest citrus producer within the European Union and the fifth worldwide with an average production of 5 million tons per season. Most of it is exported to third countries. Indeed, according to FAO, Spain is considered the first citrus exporter worldwide. Spanish citrus production is mainly destined to fresh market. Due to the high fruit quality standards demanded by this market, citrus pest management has to provide fruit with almost no pest presence or damage and at the same time, with minimal pesticide residues. International market demands place Spanish citrus industry in an unbeatable position to implement area-wide Integrate Pest Management (aw-IPM) programs. In the last years the citrus producing area that adopted IPM programs has dramatically increased. In 2012 more than 19% of the production already followed IPM regulations and that was a 5-fold increment relative to 2005. The current trend indicates that the number of growers adopting IPM systems will keep increasing over the coming years. The present article summarizes the current status of Spanish citrus IPM, how pests are hierarchized according to their biological control and which are the available IPM tools for growers. Large scale implementation of this management approach will permit Spanish citrus production to make a qualitative leap in a society that seeks for healthier eating habits and more environmental friendly production systems.

This research work presents the development of a protocol for the micropropagation of three allotetraploid somatic hybrid rootstocks, obtained at IVIA from two hybridizations: Citrus macrophylla + citrange Carrizo and C. macrophylla + C. taiwanica. We have determined the conditions for the in vitro introduction and the establishment of micropropagation lines in the laboratory, with a high efficient multiplications rate of 3 every two weeks. The environmental conditions for the successful acclimatization and growth in the greenhouse have also been established. Finally, we have accomplished the large-scale adaptation for efficient commercial propagation.

In the Mediterranean area, citrus fruits are primarily produced for the fresh fruit market and Spain is the principal producer of the area. Seedlessness is one of the most important characteristics for mandarin fresh fruits. Triploid hybrids allow implementing this trait in commercial cultivars. In this work we described 'Alborea' and 'Albir', two new triploid hybrids originated from the IVIA triploid breeding program, which will be soon released to growers. 'Alborea' was obtained from an open pollinated 'Fortune' mandarin. Fruits reach optimum maturity at the beginning of January, although they can be harvested from December until the end of January. Fruits are easy-to-peel, with a diameter of 50-60 mm, and rind is deep orange red in color. 'Albir' was obtained from a cross between 'Fortune' and 'Kara' mandarins. Fruits reach optimum maturity at the beginning of February, although they can be harvested from January until second half of February. Fruits are easy-to-peel with a diameter of 60-70 mm and fruit rind is orange red in color. This variety has the same origin as 'Safor', a previously released triploid hybrid. The fruits of the two varieties are somewhat similar, but 'Albir' matures one month earlier. With these cultivars, 'Albir' and 'Safor', it will be possible to supply similar fruits to the market from January to the end of March.

Citrus cultivars must meet two basic conditions for fresh consumption: i) they should cover the widest possible commercial calendar, and ii) allow continuous adaptation to consumer preferences. For citrus growers, this requires permanent adoption of new technology and changes in the cultivars grown, driven by income-risk criteria, as well as by technical and commercial experience. The duration of an investment depends on the agronomic performance of the cultivar or its commercial life, which may be shortened due to lack of consumer acceptance. The survival of a cultivar depends on many variables: technical, quality, and competitiveness of the cultivar. Spanish citrus production falls into five main groups of cultivars: lemons (10.8%), clementines (25%), mandarin hybrids (8.7%), navel oranges (35%) and common oranges (15%). The most productive cultivars are ‘Fino’ lemon, ‘Navelina’ navel, ‘Clemenules’, ‘Lane Late’ navel and ‘Valencia’ sweet orange. In relation to changes in varietal composition, it is of interest the introduction of new cultivars of the clemenules group (‘Orogrande’, ‘Nulessin’), navel (‘Powell’, ‘Rohde’, ‘Barnfield’, ‘Chislett’ and ‘Fukumoto’) and Valencia (‘Barberina’, ‘Midknight’, ‘Valencia Late’ Frost and ‘Delta Seedless’). There is constant pressure to supply new early clementines cultivars, but their survival is usually short. Currently, mandarin hybrids represent a group with a strong potential to expand.

Autocidal control of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), has become the cornerstone of area-wide integrated pest management programs in many countries. In this technique a critical point is the mating success of released sterile males which should contribute to reduce wild populations. Till this moment the evaluation of mating success is not performed regularly in area-wide programs, being used an indirect measure as the recapture ratio of sterile versus fertile males, to evaluate the success of the sterile insect technique. We present here a review of the state of the art in mating success evaluation including the application of a molecular tool, which allows the quantification of mating success of released sterile males, linked to the progeny reduction at different sterile males release ratios using different medfly hosts. Implications of these results on the efficacy measurement of an area-wide SIT program are discussed.

Three paraben sodium salts (p-hydroxy-benzoate) were evaluated as postharvest treatments to control citrus green (GM) and blue (BM) mold, caused by Penicillium digitatum and Penicillium italicum, respectively. The curative activity of sodium methyl (SMP; at 0.1, 1, 10, 40, 70, 100, 150, 200 and 250 mM), ethyl (SEP; at 0.1, 1, 10, 20, 30, 40, 70, and 100 mM) and propyl (SPP; at 0.1, 1, 4, 7, 10, and 100 mM) paraben were evaluated on ‘Valencia’ oranges by inoculating the pathogens in rind wounds and, about 24 h later, placing a drop of the solutions in the same rind wound. Disease development was assessed on fruit incubated at 20°C for 6 days. All three paraben salts reduced mold development. Concentrations of 150, 200 and 250 mM of SMP were the most effective and completely controlled both diseases. Concentrations of 20, 30, 40, 70 and 100 mM of SEP reduced the incidence of GM and BM by 100, 94, 100, 100 and 73% and 87, 62, 93, 100 and 80%, respectively. Concentrations of 7, 10 and 100 mM of SPP reduced the incidence of GM and BM by 94, 89 and 94% and 70, 88 and 100%, respectively. Most of these concentrations of SMP, SEP and SPP also significantly reduced the severity of GM and BM. SMP at 200 mM, SEP at 70 mM and SPP at 100 mM were, among the range of concentrations tested, selected as the best to be used in further research.

In citrus, the use of rootstocks promotes productivity, improves fruit quality and may confer resistance or tolerance to biotic and abiotic stress. ‘Rangpur’ lime (Citrus limonia, Osbeck) is one of the most tolerant rootstock to drought and is largely used by the Brazilian citrus industry. Previous studies have shown that, when grafted with a variety citrus doubled diploid (4x) ‘Rangpur’ lime rootstock is more tolerant to drought stress than the respective diploid (2x). In the present study, we characterized the water deficit tolerance in 2x and 4x ‘Rangpur’ lime seedlings. Water deficit was applied respectively for 21 days, followed by recovery irrigation. Physiological and biochemical parameters were measured periodically during the experiment and samples were collected for biochemical analysis. Doubled diploid seedlings were showed to be more water deficit tolerant than 2x. Water deficit caused a greater reduction in stomatal conductance in 2x compared to 4x. The 21st day of stress, leaf water potential in 2x was lower. Analyses of the activity of enzymes involved in detoxification process suggest that the better tolerance of 4x genotypes may be related to better scavenging of reactive oxygen species.

Production of seedless citrus fruits is required for the fresh market because consumers do not accept seedy fruits. Development of new seedless mandarin cultivars has a high priority for many citrus industries worldwide. The recovery of triploid hybrids is the most promising approach to achieve this goal, since triploids have a very low pollen and ovule fertility. They are seedless or produce very low numbers of seeds and do not induce the formation of seeds in other cultivars by cross pollination. In Spain we are carrying a triploid breeding program since 1996 based on 2x × 2x, 2x × 4x and 4x × 2x sexual hybridizations followed by embryo rescue and flow cytometry, together with the use of different approaches to produce new tetraploid parents. The objective is to obtain new high quality easy peeling and seedless mandarin cultivars. More than thirty spontaneous autotetraploid apomictic genotypes to be used as male parents have been selected from seedbeds by flow cytometry. Also nine autotetraploid non apomictic genotypes, mainly to be used as female parents, have been obtained by chromosome duplication with treatments of micrografted shoot tips with colchicine or oryzalin. Symmetric protoplast fusion is also being used to produce allotetraploid genotypes. So far, we have obtained more than 5,500 triploid hybrids from 130 parental combinations by 2x × 2x pollinations, more than 4,300 triploid hybrids from 100 parental combinations by 2x × 4x pollinations, and more than 5,600 triploid hybrids from 103 parental combinations by 4x × 2x pollinations. In 2008 we released the first cultivars originated in the program, the seedless late maturing mandarins ‘Garbí’ and ‘Safor’, obtained from 2x × 2x crosses. More than 400,000 trees of these cultivars have been planted by growers during the last three years.

Many adult parasitoids depend on sugar-rich foods such as nectar and honeydew to meet their energy requirements. Here, we first use HPLC (high performance liquid chromatograph) analyses to assess whether Aphytis melinus DeBach (Hymenoptera: Aphelinidae), the most successful biological control agent of California red scale Aonidiella aurantii Maskell (Hemiptera: Diaspididadae), feeds on honeydew in the field. Our data showed that A. melinus, whose host does not produce honeydew, fed commonly on honeydew from other abundant hemipterans in spring and summer. In a second assay, we studied the longevity of A. melinus when it had access to honeydew excreted by five common hemipterans in citrus: Aleurothrixus floccosus (Maskell) (Aleyrodidae), Aphis spiraecola Patch (Aphididae), Coccus hesperidum L. (Coccidae), Icerya purchasi Maskell (Monphlebidae) and Planococcus citri (Risso) (Pseudococcidae). A. melinus longevity entirely depended on the honeydew source. The longevity of females with access to honeydew excreted by A. spiraecola was similar to unfed females. Contrarily, C. hesperidum and I. purchasi excreted the honeydew with highest nutritional value.

Citrus psorosis virus (CPsV), genus Ophiovirus, causes an important disease in many countries. In growing regions where natural disease spread occurs, damage caused by the virus can be controlled using resistant or tolerant cultivars, but sensitivity of many species and hybrids of Citrus and related genera is presently unknown. To find potential sources of CPsV resistance, we first propagated 61 species (or cultivars) and hybrids of Citrus and related genera [Citrus (37), Microcitrus (5), Fortunella (6), Eremocitrus (1), Pleiospermium (1), Atalantia (1), Severinia (1), Clausena (1), Poncirus (1) and hybrids (7)] on Rough lemon seedlings inoculated with the CPsV isolate PB 143. Out of the 61 accessions tested 54 showed symptoms and reacted by ELISA with a CPsV-specific antibody, 2 (M. inodora and F. hindsii) were symptomless but gave high ELISA values, suggesting tolerance to CPsV, 5 (C. depresa, Cleopatra mandarin, C. excavata, Carrizo citrange and CPB 4475 citrumelo) showed symptoms in the first flush but gave a negative ELISA reaction, and 1 (Poncirus trifoliata) was ELISA negative and symptomless. We then examined CPsV infection by ELISA and RT-PCR in Cleopatra mandarin, P. trifoliata and Citrumelo seedlings inoculated with CPsV isolates P 121, PB 108 and PB 143. While P 121 was detected in all Cleopatra and citrumelo plants, the other isolates were detected in only 50% of the inoculated plants, with all plants indexed as CPsV positive displaying symptoms. All P. trifoliata plants inoculated with either isolate indexed negative for CPsV and remained symptomless. Our results suggest CPsV resistance in P. trifoliata and partial isolate-specific resistance in some accessions.

Polyploidy has long been recognized as an important force in the evolution of flowering plants and often results in novel phenotypes having advantages in adaptation and major interest for agriculture. Many crops, including citrus, are bred to a higher level of ploidy in search of desirable traits. In previous works of our group synthetic clementines with different levels of ploidy, specifically haploid, dihaploid and autotetraploid, were generated. This plant material represents a valuable tool for studying the effects of polyploidization at the molecular level since important traits are expressed in different manners. By using a microarray approach, we have accomplished a comprehensive analysis of transcriptome divergence among the newly created clementine lineages with different genome dosage. Differences in expression in the bark tissue of the four lineages were found significant for 89 (haploid-dihaploid comparison) or 93 genes (diploid-tetrapolid comparison). These genes were classified on the basis of their structure in different families.

Citrus trees, traditionally associated with Spanish Mediterranean agriculture, are particularly sensitive to water allocation fluctuations. In this area structural water resource deficits, as well as the gradual decline in citrus prices received by farmers, have promoted the co-plantation of other crops that are more resistant to water shortages, such as pomegranates, which have been grown in eastern Spain for decades. At present, demand for pomegranates is growing due to their associated nutritional benefits and functional food qualities. In this paper, we have analyzed technical-economic aspects of citrus and pomegranate cultivation in light of their increasing competition. The comparison of these two crops begins with an assessment, from a farm economy perspective, of the main results published in the scientific literature on Regulated Deficit Irrigation, which is one of the techniques used to address water shortage problems. Also, we have analyzed the trend in prices received by farmers for the orange ‘Lane late’, the mandarin ‘Clemenules’ and for the two main Spanish varieties of pomegranate, ‘Valenciana’ and ‘Mollar’. The economic outlook for these two crops (citrus and pomegranate) is rounded off by an investment analysis, which describes the profitability of individual farms.

A triploid breeding program has been carried out in Plant Protection and Biotechnology Center at the Instituto Valenciano de Investigaciones Agrarias (IVIA) based on sexual hybridization, embryo rescue and ploidy analysis by flow cytometry in order to obtain new mandarin cultivars with late harvest. 'Safor' and 'Garbi' mandarins are new triploid hybrids recently released characterized by their high quality and seedless fruits. In the Citrus Center and Vegetable Production (IVIA) another breeding program based on budwood irradiation is being conducted in search of new seedless cultivars. At this moment two clones obtained from irradiated budwood of the seedy 'Moncada' mandarin (Moncalina-6S17 and Manrad-2L09) are in process of being registered and exhibit excellent fruit quality. This study aimed at testing the postharvest behavior of these four new cultivars. After harvest the fruit were waxed and stored up to 30 days at 1, 5, or 9 degrees C in order to evaluate the sensibility to chilling injury and the changes in quality parameters that occur during the cold storage. 'Safor' mandarins did not show chilling injury symptoms during 30 days at any temperature studied. In contrast, 'Garbi' mandarins exhibited susceptibility to chilling injury after 15 days of storage at 1 and 5 degrees C; during this period this cultivar showed disorders related with peel dehydration when stored at 9 degrees C. 'Moncalina' and 'Manrad' did not show relevant symptoms related to cold-induced damage or dehydration at 1, 5 and 9 degrees C for 30 days of storage. For all mandarin cultivars assayed, the storage at low temperature was not limited for the changes observed in fruit quality parameters.

Despite considerable differences in morphology, the genera representing 'true citrus fruit trees' are sexually compatible, but their phylogenetic relationships remain unclear. Most of the important commercial species of Citrus are believed to be of interspecific origin. By studying SNP and indel polymorphisms of 27 nuclear genes on 32 genotypes of Citrus and relatives of Citrus, the phylogenetic relationship between 'true citrus fruit trees' was clarified. Additionally, four mitochondrial (mtDNA) and five chloroplastic (cpDNA) SSRs markers were analysed for the citrus species to specify the maternal phylogeny of Citrus secondary species. A total of 16238 kb of DNA was sequenced for each genotype, and 1156 SNPs and 58 indels were identified. Nuclear phylogenetic analysis revealed that C. reticulata, Poncirus, and Fortunella form a clade that is clearly differentiated from the clade that includes three other basic taxa of cultivated citrus (C. maxima, C. medica and C. micrantha). Clymenia is included in a third strong clade with Eremocitrus and Microcitrus. The nuclear phylogeny of Citrus and its sexually compatible relatives was consistent with the geographic origins of these genera. The origin of Citrus secondary species was analysed both at nuclear and cytoplasmic level and the conclusions mostly agree with previous hypotheses. The SNPs and indels identified at the nuclear level from a relatively large discovery panel will be useful for systematic characterisation of citrus germplasm, both at intra-and inter-specific level.

The citrus nursery tree certification program in Spain includes four blocks of trees: 1) The protected foundation block, maintained at IVIA, that includes healthy plants of local and foreign varieties recovered by shoot-tip grafting in vitro. Plants are grown in containers inside insect-proof screenhouses. 2) Foundation blocks propagated with budwood from the protected foundation block and also grown inside screenhouses. They belong to individual nurseries or nursery groups. 3) Budwood increase blocks, maintained inside screen-or greenhouses at each nursery, include plants propagated directly from foundation trees to increase the number of buds for propagation of certified trees. 4) Certified nursery trees propagated with budwood from the increase blocks and grown in screen-or greenhouses or in the open field. Plants of each block are periodically indexed for different pathogens according to the regulations. The program started in 1979 when the first healthy plants were released to the nurseries and the first certified plants arrived to growers in 1982. Since then, more than 142 million certified plants of 103 varieties have been propagated, with sweet oranges representing 51%, clementines 28%, other mandarins 14%, lemons 6% and grapefruits 1%. Presently Carrizo citrange is used as rootstock for 59% of the nursery plants and Citrus macrophylla for 19%. A total of 40 nurseries, grouped in 7 foundation blocks, are presently operating. Under this program practically all the Spanish citrus industry has been renewed with healthy plants. Today, traditional graft transmissible pathogens are not a problem for our citrus industry.

The endoparasitoid Diachasmimorpha longicaudata (Ahsmed) (Hymenptera: Braconidae) is one of the larval parasitoids used in biological control of Tephritid fruit flies. Ceratitis capitata (Diptera: Tephritidae) is an economically important fruit fly of special importance in Spain, and mainly in the Valencia Community by the economic importance of the citrus sector. Nowadays the control of C. capitata in Valencia is based on the integration of area-wide Sterile Insect Technique (SIT) program with pest monitoring, mass trapping and chemical applications. The biological control of C. capitata is under study, for this reason in 2009 the larval parasitoid D. longicaudata was imported from Mexico. A release study is presented here to determine its contribution to medfly control. Different densities of D. longicaudata adults were released once each season in a citrus plot to determine its dispersion on the plot and the parasitism rate exerted in infested sentinel apples. The results showed that the parasitoid D. longicaudata is able to disperse and parasitize C. capitata larvae in Mediterranean climatic conditions.

Postharvest degreening treatment with exogenous ethylene application is widely used in Mediterranean growth area to improve the external coloration of the early-season citrus varieties in which internal maturity is achieved when the peel is still green. Nevertheless this treatment can induce physiological disorders overall related to calyx senescence. In previous studies it has been reported that the application of 3,5,6-trichloro-2-pyridyloxyacetic acid (TPA) reduces calyx senescence associated to ethylene exposure. However it is important to know the effect of this auxin under commercial industrial conditions. The purpose of this study was to evaluate the effect of postharvest application of TPA on the reduction of disorders associated to degreening in clementines in different commercial post-harvest applications (by drencher and by low volume spraying in the handling line). The application of TPA by drencher significantly reduced the disorders of calyx senescence associated to degreening treatment in clementines, recommending 40 ppm of TPA as the most effective dose. The application of TPA by low volume spraying in the handling line treatments was effective on the reduction of calyx disorders, obtaining the best results with 100 ppm of TPA; nevertheless in the trials clementines exhibited low susceptibility to calyx disorders after degreening process. The results suggest that this auxin could be a good option to control disorders of citrus fruit during degreening treatment.

The phytosanitary surveillance plays an important role in the early detection to prevent the establishment of harmful pests of economic and quarantine importance. The presence of fruit flies belonging to the genera Anastrepha, Bactrocera or Ceratitis (Diptera: Tephritidae) on the American continent constitute a potential risk of introduction, threatening the production of citrus and other hosts of countries in the region. In Cuba, the presence of some Anastrepha spp., as well as Bactrocera carambolae or Ceratitis capitata is not registered, thus surveillance for these quarantine pests and for other species of national interest continues. Results have been obtained from research on preferred host fruits located in the surroundings of citrus plots, revealing the presence and potential risk to citrus of A. suspensa, identified as of quarantine importance in countries such as the United States and others. During the period 2010-2012 three activities were conducted to characterize, diagnose and train staff to strengthen phytosanitary surveillance of Anastrepha fruit flies in citrus production areas in: Ceiba Citrus Enterprises (Artemisa), Victoria de Giron (Matanzas), Arimao (Cienfuegos) and Ciego de Avila citrus enterprises. To fulfill this objective, the composition of host fruit presence in production areas with respect to A. suspensa was evaluated. Host preferences of the main Anastrepha species detected and the effectiveness of traps and attractants were determined, and the monitoring system was relocated. The information obtained allowed designing the monitoring strategy in production areas. Training, one of the most important tools, was developed through seminars and workshops. These results provide inputs to the monitoring programs and the procedures that are essential in the development and implementation of measures for the phytosanitary surveillance and integrated management of fruit flies in Cuban citrus production.

Psorosis disease of citrus is characterized by bark scaling in the stem and main branches of trees at least 10 years old. Graft-inoculation of sweet orange (Citrus sinensis (L.) Osb.) seedlings in the greenhouse with non-scaled bark pieces from an infected tree incites the psorosis A syndrome (PsA), including a shock reaction of the first flush and transient chlorotic flecking in young leaves of the following flushes, whereas inoculation with scaled bark pieces incites the psorosis B syndrome (PsB), that in addition to PsA symptoms includes gummy pustules in old leaves and branches. Psorosis disease is presumably caused by Citrus psorosis virus (CPsV), an ophiovirus with three negative-stranded genomic RNAs. While comparison of RNA1 or RNA3 fragments by single-strand conformation polymorphism (SSCP) analysis did not show any difference between plants affected by the PsA or the PsB syndromes, a fragment of RNA2 enabled discriminating both syndromes by their SSCP profile. A plant inoculated with scaled bark did not show PsB at 6 months post-inoculation (mpi) and its RNA2 gave a PsA-type SSCP profile. At 12 mpi this plant showed pustules in the trunk but not in the leaves. SSCP analysis of RNA2 showed only PsA-type variants in the leaves, whereas the pustuled trunk areas contained both PsA and PsB variants, the latter being predominant. These results suggest that psorosis-affected trees contain the two sequence variants and that the PsB variant is associated with pustules and tends to accumulate in the trunk bark.

The citrus aphids Aphis spiraecola Patch. and Aphis gossypii Glover (Hemiptera: Aphididae) are key pests of citrus clementine in Spain. A rich complex of natural enemies (NE) exploits these two species. However, NE usually colonize citrus trees once aphid populations have exceeded economical thresholds. Hence, successful biological control should be based on anticipating the arrival of NE. Recently, our group has found that citrus orchards with a ground cover of grass (Poales: Poaceae) had less aphids per colony and these colonies lasted shorter than those colonies found in orchards with bare soil management. These differences were attributed to an earlier appearance of aphid NE in the grass cover which attracted NE to the citrus orchard. This early appearance of NE contributed to a successful control of aphids in the canopy.

A proteomic study was used to know how the primary metabolism in Citrus leaves is affected by the fruit load. To this end, we researched the differential expression of proteins related to these processes between on-crop and off-crop ‘Moncada’ mandarin leaves. Samples were collected in November and proteins were extracted. From 2D DIGE gel of these extracts, 33 spots related to primary metabolism were isolated: 26 spots being associated with photosynthesis and carbohydrate metabolism, 4 spots related to Krebs cycle, 1 spot related to pentose phosphate pathway, and 2 spots related to nutrient reservoir activity. These spots were identified by MALDI-MS or LC-MS-MS. Between the proteins related to photosynthesis and carbohydrate metabolism, 16 were up-expressed in off-crop leaves, such as NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, RuBisCO large subunit, ADP-glucose pyrophosphorylase, cinnamoyl-CoA reductase, and carbonic anhydrase; while 10 proteins were up-expressed in on-crop leaves such as NADP-dependent malic enzyme, and 3,4-dihydroxy-2-butanone kinase. The isolated proteins related to pentose phosphate pathway (6-phosphogluconate dehydrogenase) and to nutrient reservoir activity (Granule-bound starch synthase Ib precursor) were up-expressed in off-crop leaves. Regarding the proteins related to Krebs cycle, only malate dehydrogenase was up-expressed in off-crop leaves, while the other proteins were up-expressed in on-crop leaves (citrate synthase and NADP-isocitrate dehydrogenase). According to these results, the primary metabolism seems to be more active in off-crop leaves, suggesting that fruit load inhibits the primary metabolism in ‘Moncada’ mandarins.

Alternaria brown spot (ABS) in citrus is a serious disease caused by the tangerine pathotype of the fungus Alternaria alternata, which induces necrotic lesions on fruit and young leaves, defoliation and fruit drop in susceptible genotypes. It is a strong concern for triploid breeding programs aiming to produce seedless mandarin cultivars. In the present work, we have taken advantage of the particular genetic structures of unreduced megagametophytes resulting from second division restitution (SDR) (leading to triploid hybrid recovery) to map a genome region linked to ABS resistance in citrus. The monolocus dominant inheritance of susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. Bulk segregant analysis coupled with genome scan using a large set of genetically mapped SNP markers and targeted genetic mapping by half tetrad analysis, using SSR and SNP markers, allowed locating a region linked to ABS resistance near the centromere of chromosome III. SSR and SNP markers were developed for efficient early marker-assisted selection (MAS) of ABS resistant hybrids.

Alternaria brown spot (ABS) of mandarins, caused by a pathotype of the fungus Alternaria alternata, is a serious disease both in humid and semi-arid citrus-growing regions of the world. The pathogen affects leaves and fruit of susceptible mandarin cultivars such as 'Fortune', 'Nova', 'Minneola', and 'Murcott'. The epidemiology of ABS was studied mainly in humid areas in Florida but, due to climatic differences, this information cannot be extrapolated to semi-arid regions like Spain. Field studies were conducted in 2011 in the experimental orchards at IVIA, Valencia. Dynamics of the airborne Alternaria conidia were monitored weekly by a spore trap, and the percentage of pathogenic isolates was determined periodically using the selective medium, ARSA, and pathogenicity tests. The presence of inoculum on affected leaves, shoots, leaf litter, and weeds was also determined. Infection periods were monitored weekly by exposing trap or indicator plants of 'Fortune' and 'Nova' mandarins. Environmental variables were recorded by an automated meteorological station. Although Alternaria conidia were detected through the experimental period, only 5% of all the isolates were pathogenic. As affected leaves and shoots were determined to be the main source of inoculum, the survival of the fungus in the leaf litter was higher than previously reported in Florida. Pathogenic isolates were detected also in weeds, but only at very low levels. Infections in trap plants occurred mainly in May-June and September-October. A significant positive correlation was detected between disease incidence and rainfall.

Tetranychus urticae Koch (Acari: Tetranychidae) populations in citrus can be regulated through the management of the cover crop. A mono-specific cover of the grass Festuca arundinacea Schreber (Poaceae) enhanced the diversity and abundance of Phytoseiidae mites compared with a multifloral cover. This increase resulted in a better control of T. urticae. Pollen availability and quality could be behind this result. Whereas multifloral cover offers a long-term source of a wide variety of pollen, F. arundinacea offers one single type of pollen in spring only. As a consequence, the generalist pollen feeder Euseius stipulatus (Athias-Henriot), which is a superior intra-guild predator, is highly favored in multifloral covers. Therefore, poor quality pollen may prevent pollen feeders from reaching high numbers and indirectly benefit T. urticae control by specialist phytoseiids.

Tetranychus urticae Koch (Acari: Tetranychidae) is a key pest of clementine mandarins, Citrus clementina Tanaka (Rutaceae), in Spain. This mite is highly polyphagous and can be easily found in clementine orchards, both in the trees and in the associated flora. In a previous study we found that the use of a cover of Festuca arundinacea Schreber (Poaceae) offered a better regulation of T. urticae populations than either bare soil or the traditional wild cover, which included a mix of weed species. We hypothesized that the selection of two host races of T. urticae, specialized in F. arundinacea and C. clementina, could partly explain the results obtained in field studies (bottom-up regulation). Reciprocal transplant experiments show that sympatric deme x host combinations had higher mean fitness values for most of the parameters evaluated than the allopatric combinations in clementine, but not in F. arundinacea. Because local adaptation implies mean deme fitness to be systematically higher for the sympatric deme x habitat combinations than for the allopatric ones, these results can be taken as indicative of occurrence of local adaptation in T. urticae. Molecular genetic analyses with microsatellite markers support this conclusion and indicate that host adaptation of T. urticae found in our system may indeed contribute to a better natural regulation of this mite.