DRIS norms for 'Valencia' sweet orange on three rootstocks

Diagnosis and Recommendation Integrated System (DRIS) applies nutrient ratios instead of the isolated concentration values of each nutrient in interpretation of tissue analysis. The objectives of this research were to establish adequate DRIS norms for 'Valencia' sweet orange irrigated commercial groves budded on three rootstocks and correlate indexes of nutrition balance with yield. Experiments were conducted in São Paulo State, Brazil. Rootstocks Rangpur lime, Caipira sweet orange, and Poncirus trifoliata, with more than six years old and yield above 40 ton ha-1 were utilized. Data referred to yield, tree spacing, rootstock and foliar concentrations of N, P, K, Ca, Mg, S, Fe, Mn, Cu, Zn, and B in non fruiting terminals for each grove were processed for the years 1994 through 1998. DRIS indexes were calculated by Nick criterion for choosing the ratio order of the nutrients and Jones calculation method of the ratio functions. Indexes of nutritional balance calculated from DRIS norms presented high correlation with yield for the three scion/rootstock combinations. DRIS norms defined in this research are valid, since leaf sampling is done on non fruiting terminals and the grove is irrigated.

Sweet orange trees grafted on selected rootstocks fertilized with nitrogen, phosphorus and potassium

The majority of citrus trees in Brazil are grafted on 'Rangpur lime' (Citrus limonia Osb.) rootstock. Despite its good horticultural performance, search for disease tolerant rootstock varieties to improve yield and longevity of citrus groves has increased. The objective of this work was to evaluate yield efficiency of sweet oranges on different rootstocks fertilized with N, P, and potassium. Tree growth was affected by rootstock varieties; trees on 'Swingle' citrumelo [Poncirus trifoliata (L.) Raf. × C. paradisi Macf.] presented the smallest canopy (13.3 m³ in the fifth year after tree planting) compared to those on 'Rangpur lime' and 'Cleopatra' mandarin [C. reshni (Hayata) hort. ex Tanaka] grown on the same grove. Although it was observed an overall positive relationship between canopy volume and fruit yield (R² = 0.95**), yield efficiency (kg m-3) was affected by rootstocks, which demonstrated 'Rangpur lime' superiority in relation to Cleopatra. Growth of citrus trees younger than 5-yr-old might be improved by K fertilization rates greater than currently recommended in Brazil, in soils with low K and subjected to nutrient leaching losses.

Stability of Citrus tristeza virus protective isolate 'Pêra IAC' according to SSCP analysis of old and new lines of three sweet orange varieties

Clonal cleaning, followed by pre-immunization with protective complexes of Citrus tristeza virus(CTV), allowed the commercial cultivation of Pêra sweet orange, a variety that has great importance for Brazilian citriculture but is sensitive to the virus. The use of mild protective isolates in other citrus varieties, even those more tolerant to CTV, can also be of interest to prevent the spread of severe isolates. The aim of this study was to characterize, by means of SSCP (Single Strand Conformational Polymorphism) analysis of the coat protein gene, CTV isolates present in plants of the sweet orange cultivars Pêra, Hamlin and Valencia propagated from four budwood sources: 1) old lines, 2) nucellar lines, 3) shoot-tip-grafted lines, and 4) shoot-tip-grafted lines pre-immunized with the mild CTV protective isolate 'PIAC'. We also evaluated the correlation of the obtained SSCP patterns to stem pitting intensity, tree vigor and fruit yield. SSCP results showed low genetic diversity among the isolates present in different trees of the same variety and same budwood source and, in some cases, in different budwood sources and varieties. Considering tristeza symptoms, lower intensity was noted for plants of new, shoot-tip-grafted and pre-immunized shoot-tip-grafted lines, compared to old lines of the three varieties. The observed SSCP patterns and symptomatology suggested that more severe CTV complexes infect the plants of old lines of all three varieties. The protective complex stability was observed in the SSCP patterns of CTV isolates of some shoot-tip-grafted and pre-immunized clones. It was concluded that the changes detected in other electrophoretic profiles of this treatment did not cause loss of the protective capacity of CTV isolate 'PIAC' inoculated in the pre-immunization.

Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange

The development and optimization of efficient transformation protocols is essential in new citrus breeding programs, not only for rootstock, but also for scion improvement. Transgenic 'Hamlin' sweet orange (Citrus sinensis (L.) Osbeck) plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated in vitro. Factors influencing genetic transformation efficiency were evaluated including seedling incubation conditions, time of inoculation with Agrobacterium and co-culture conditions. Epicotyl segments were adequate explants for transformation, regenerating plants by direct organogenesis. Higher percentage of transformation was obtained with explants collected from seedlings germinated in darkness, transferred to 16 hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45 min. The best co-culture condition was the incubation of the explants in darkness, for three days in culture medium supplemented with 100 muM of acetosyringone. Genetic transformation was confirmed by performing beta-glucoronidase (GUS) assays and, subsequently, by PCR amplification for the nptII and GUS genes.

Genetic linkage maps of 'Pêra' sweet orange and 'Cravo' mandarin with RAPD markers

The objective of this work was to construct linkage maps of 'Pêra' sweet orange [Citrus sinensis (L.) Osbeck] and 'Cravo' mandarin (Citrus reticulata Blanco) using RAPD markers and the pseudo-testcross strategy. The parents were chosen according to the resistance/susceptibility to citrus variegate chlorosis (CVC). The segregation of 176 markers was analyzed in 94 progeny of F1 hybrids, which were obtained from controlled crossings. The linkage map of 'Pêra' sweet orange had 117 markers defined by 12 linkage groups, which spanned 612.1 cM. Only six markers could not be linked to the linkage group and 48.7% of the markers showed segregation distortion. The linkage map of 'Cravo' mandarin had 51 markers defined by 12 linkage groups, which spanned 353.3 cM. Only two markers did not link to the groups and 15.7% showed segregation distortion. The construction of linkage maps is relevant to future mapping studies of the inheritance of CVC, citrus canker and leprosis.

GUS gene expression driven by a citrus promoter in transgenic tobacco and 'Valencia' sweet orange

The objective of this work was the transformation of tobacco and 'Valencia' sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL) gene promoter (CsPP). Transformation was accomplished by co-cultivation of tobacco and 'Valência' sweet orange explants with Agrobacterium tumefaciens containing the binary vector CsPP-GUS/2201. After plant transformation and regeneration, histochemical analyses using GUS staining revealed that CsPP promoter preferentially, but not exclusively, conferred gene expression in xylem tissues of tobacco. Weaker GUS staining was also detected throughout the petiole region in tobacco and citrus CsPP transgenic plants.

Tissue culture parameters in sweet orange cultivars

The objective of this work was to establish tissue culture parameters for gene transfer in sweet orange cultivars. Epicotyl explants with different ages were cultured with 6-benzylaminopurine (BAP), kanamycin and hygromycin. Shoots were cultured with alpha-naphthaleneacetic acid (NAA) alone or in combination with indole-3-butyric acid (IBA). The requirement of BAP for shoot development was genotype-specific. Epicotyl explants from 35-day-old seedlings produced significantly more shoots per explant in 'Pêra'. Kanamycin inhibited shoot regeneration for the most cultivars. The percentage of shoots that produced roots in 'Pêra' was significantly higher in medium with NAA and IBA than with NAA alone.

In vitro organogenesis from internodal segments of adult sweet orange plants

The objective of this work was to optimize in vitro plant regeneration via organogenesis from tissues of adult 'Hamlin', 'Pêra', and 'Valência' sweet orange plants. Explants were grown in EME culture medium with different concentrations of 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA), at 27ºC in the absence of light for 50 days, followed by a 16-hour photoperiod for 20 days. Regeneration was assessed 50 and 70 days after in vitro culture. Organogenesis in cultivars Hamlin and Valência was promoted by EME supplemented with BAP, while NAA showed no apparent effect.

Reaction of sweet orange cultivars expressing the attacin A gene to 'Candidatus Liberibacter asiaticus' infection

The objective of this work was to evaluate the reaction of four sweet orange cultivars expressing the attacin A gene to 'Candidatus Liberibacter asiaticus' (Las) infection, a bacterium associated to huanglongbing (HLB) disease. Transgenic sweet orange plants of Hamlin, Natal, Pêra, and Valência cultivars, as well as nontransgenic controls received inocula by grafting budwood sections of HLB-infected branches. Disease progression was evaluated through observations of leaf symptoms and by polymerase chain reaction (PCR) analysis, eight months after inoculation. A completely randomized design was used, with four experiments (one for each cultivar) performed simultaneously. Bacteria title was estimated by quantitative PCR (qPCR). HLB symptoms and Las titers were present in nontransgenic and transgenic plants expressing the attacin A gene of the four sweet orange cultivars, eight months after bacteria inoculation. Five transgenic lines (transformation events) of 'Pêra' sweet orange expressing the attacin A gene have significantly lower Las titers in comparison with nontransgenic plants of this cultivar.

Development of globular embryos from the hybridization between 'Pêra Rio' sweet orange and 'Poncã' mandarin

This research was undertaken to study the influence of different concentrations of the MT medium, sucrose, vitamins, activated charcoal and gibberellic acid (GA3) on the culture of immature embryos from the crossing between 'Pêra Rio' sweet orange and 'PONCÃ' mandarin. The embryos were excised under aseptic conditions and inoculated in 15 mL of the MT medium according to the following experiments: 1) MT concentrations (0%, 50%, 100%, 150% and 200%) supplemented with 0, 30, 60 and 90 g.L-1 of sucrose; 2) vitamins concentrations of the MT (0%, 50%, 100%, 150% and 200%) supplemented with 0, 30, 60 and 90 g.L-1 of sucrose; 3) activated charcoal concentrations (0, 0.5, 1, 1.5 and 2 g.L-1) supplemented with GA3 (0, 0.01, 0.1; 1 and 10 mg.L-1). After the inoculation, the embryos were kept in a growth room for 90 days at 27 ± 1ºC, in a 16-hour photoperiod with 32 µmol.m-2.s-1 of irradiance. The best development of embryos at the globular stage was achieved using 50% and 100% of the MT medium plus 60 g.L-1 and 90 g.L-1 of sucrose, respectively, supplemented with 0.01 mg.L-1 of GA3. The addition of activated charcoal or vitamins in the MT medium has shown to be unnecessary to the development of globular embryos.

Effect of prestorage curing on storage life, internal and external qualities of sweet orange (Citrus sinensis)

Orange fruits from two seasons, in April and August 2006 representing late 2005 and early 2006 harvests respectively were cured in hot air at 36-37(0)C to 1%, 3%, 5% and 7% weight loss before storage at 28(0)C and 86% relative humidity (RH). The fruits were observed for incidence of decay, further weight loss, juice content, firmness or softening of the peel, total soluble solids (TSS), pH, titratable acidity, and colour during storage. Curing reduced the incidence of decay. All control fruits were rotten by day 21 in August harvest while 22.5% of the control was rotten by day 56 in the April harvest. Storage life was extended beyond 56 days in fruits cured with 1, 3, 5 and 7% in April harvest as there was no decay throughout, while decay incidence in August harvest was 88.9, 61.1, 22.2 and 31.3% in 1, 3, 5 and 7% respectively. Penicillium digitatum, Phytophthora sp., Alternaria citri and Collectotrichum gloeosporioides were among decay causing moulds detected. Control fruits lost more weight during storage than cured fruits did. Fruit rind hardening was more noticed in the control and those cured to 1% weight loss, especially from the April harvest. It was insignificant in other treatments in both trials. Titratable acidity, pH, juice content and TSS were not affected by the treatment. Colour change to yellow was however retarded by curing. Curing to 5% weight loss was best for decay control and quality retention.

Natural colonization of leaves of 'Pêra' sweet orange and related varieties by Guignardia citricarpa

The purpose of this research was to evaluate the differences in the colonization and production of structures in the leaves of 'Pêra' sweet orange (Citrus sinensis) clones and related varieties by Guignardia citricarpa. The natural colonization and the production of reproductive structures in the leaves and in vitro of ten 'Pêra' sweet orange was quantified in the following clones: Bianchi, Dibbern C.V., EEL, IAC 2000, Olímpia 15161, Premunizada 1212, Premunizada 1743/82, R. Gullo 1569/244, R. Gullo 1570/246 and Vimusa; and in five related varieties: Redonda C.N, Ovale 968, Ovale San Lio 969, Lamb Summer and Corsa Tardia. The quantification of the colonization density of G. citricarpa in the leaves was obtained through isolation. Incidence and colonization density (cm²) were calculated for each clone. The production of reproductive structures was accomplished through the moistening and drying process of the leaves. The incidence (percentage of affected leaves) and the leaf surface percentage occupied by the reproductive fungus structures were quantified. The in vitro production of reproductive structures was accomplished in water-agar medium. The number of immature and total reproductive fungus structures (cm²), and the percentage of picnidia with liberation of spores were quantified. Significant differences were not observed among clones related to the colonization of the leaves. But there were differences in the induction experiments, i.e., in the leaf surface percentage occupied by the reproductive fungus structures and the in vitro production of reprodutive fungus structures.

Effect of temperature, leaf wetness, and rainfall on the production of Guignardia citricarpa ascospores and on back spot severity on sweet orange

The black spot of citrus (Citrus sp.) is caused by Guignardia citricarpa with ascospore production depending on temperature, leaf wetness, and rainfall. The number of ascospores produced was monitored using a spore trap and climatic factors were recorded using an automated meteorological station of 'Natal' and 'Valencia' sweet orange (Citrus sinensis) orchards in Mogi Guaçu in the state of São Paulo, Brazil, from November 2000 to March 2001. The fruits were bagged to prevent infection and the bags removed from different sets of fruit for one week during each of the 18 weeks of the season in both orchards. Ascospores were produced during the entire experimental period, from spring through summer, primarily after rain events. In both orchards, ascospore production reached a peak in January and February. Ascospore production was related to leaf wetness only in the Natal orange orchard but was not related to total rainfall or temperature in either orchard. Disease was most severe on fruit exposed the 7th, 8th, and 13th weeks after beginning the experiment in both cultivars as well as after the 16th week for 'Natal'. There was a strong relationship between disease severity and total rainfall for both orchards and a weak correlation between temperature and severity in the 'Natal' block only. There was no relationship between severity and leaf wetness or ascospore numbers.

Susceptibility of fruits of the 'Valência' and 'Natal' sweet orange varieties to Guignardia citricarpa and the influence of the coexistence of healthy and symptomatic fruits

The importance of the presence of symptomatic fruits with citrus black symptoms (CBS) on the disease severity level in subsequent crop production was assessed in an orchard planted with Valência and Natal sweet orange varieties. Additionally, the period of susceptibility of the fruits of these varieties was evaluated. Fruits were covered with paper bags at the stage of 75% fallen petals and were then exposed to natural infection at weekly intervals, from October 2000 to April 2001. This process was carried out in plants where the fruits from the previous harvest had been picked as well as in plants where fruits remained until natural drop. The evaluation of disease severity used a scale that varied from 0 (absence of symptoms) to 6 (severe symptoms). It was observed that, for the Valência and Natal varieties, conidia of Phyllosticta citricarpa that had formed on the lesions of fruits from the previous harvest did not significantly increase the severity of disease on the fruits of the subsequent harvest period. In this study, the protection of the fruits until 10 weeks after petal drop did not affect the number of lesions, indicating that ascospore discharges after that date were, probably, responsible for disease severity. Fruits exposed between the 20th and 24th week after 75% of the petals had fallen were symptomatic, indicating that, at this stage, the fruits were still susceptible to the pathogen.

Effect of trickle irrigation on root development of the wet bulb and 'pera' orange tree yield in the state of São Paulo, Brazil

The aim of this study was to evaluate the effect of different microirrigation designs on root system distribution in wet bulb region, orange orchard yield and quality of orange fruits. The experiment was installed as random blocks with five treatments and four replicates in an orchard of 'Pêra' orange trees grafted on 'Cleopatra' mandarin rootstock. The treatments consisted of: one drip line (T1), two drip lines (T2), four drip lines (T3) per planting row, microsprinkler irrigation (T4) and without irrigation (T5). Irrigation treatments favored yield and ºBrix. The treatment with a single drip line (T1) showed the greatest quantity of roots in relation to the treatments T2 and T3.