Histopathology of black spot symptoms in sweet oranges

In Brazil, citrus black spot (CBS) caused by Guignardia citricarpa is a major disease that has different symptoms on fruit. In this study, fruit of Citrus sinensis infected by G. citricarpa and showing the symptoms false melanosis, freckle spot and hard spot were cross-sectioned and analysed anatomically and histochemically by light microscopy. Immuno-histological assays were performed. All symptoms were accompanied by a thickening of the cuticle. False melanosis lesions did not contain pycnidia and remained restricted to the epicarp or to the first layers of the mesocarp. The stomata in this type of lesion showed phenolic compounds in the guard cells and in the sub-stomatal chamber. In some samples, the guard cells and their surrounding cells lysed, and a wound meristem began to form underneath them. Freckle spot and hard spot lesions had very similar histological alterations to the epicarp and mesocarp, but in our samples only hard spot lesions contained pycnidia. Both of these symptoms were accompanied by protein inclusions. Epidermal and sub-epidermal cells located in the oil-gland region were obliterated, causing alterations in these structures. All symptoms had regions that stained strongly for lipids and phenols.

Genetic transformation of Citrus sinensis with Citrus tristeza virus (CTV) derived sequences and reaction of transgenic lines to CTV infection

Transgenic Citrus sinensis (L.) Osb. plants, cvs. Valencia and Hamlin, expressing Citrus tristeza virus (CTV) derived sequences were obtained by genetic transformation. The gene constructs were pCTV-CP containing the 25 kDa major capsid protein gene (CTV-CP), pCTV-dsCP containing the same CTV-CP gene in an intron-spliced hairpin construct, and pCTV-CS containing a 559 nt conserved region of the CTV genome. The transgenic lines were identified by PCR and the transgene integration was confirmed by Southern blot. Transgene mRNA could be detected in most transgenic lines containing pCTV-CP or pCTV-CS transgene. The mRNA of pCTV-dsCP transgene was almost undetectable, with very light bands in most analyzed plants. The transgene transcription appears to be closely linked to the type of gene construct. The virus challenge assays reveals that all transgenic lines were infected. However, it was possible to identify propagated clones of transgenic plants of both cultivars studied with a low virus titer, with values similar to the non-inoculated plants (negative control). These results suggested that the transgenic plants present some level of resistance to virus replication. The higher number of clones with low virus titer and where mRNA could not be detected or was presented in a very light band was found for pCTV-dsCP-derived transgenic lines.

Seasonal effects on the relationship between photosynthesis and leaf carbohydrates in orange trees

To understand the effect of summer and winter on the relationships between leaf carbohydrate and photosynthesis in citrus trees growing in subtropical conditions, 'Valencia' orange trees were subjected to external manipulation of their carbohydrate concentration by exposing them to darkness and evaluating the maximal photosynthetic capacity. In addition, the relationships between carbohydrate and photosynthesis in the citrus leaves were studied under natural conditions. Exposing the leaves to dark conditions decreased the carbohydrate concentration and increased photosynthesis in both seasons, which is in accordance with the current model of carbohydrate regulation. Significant negative correlations were found between total non-structural carbohydrates and photosynthesis in both seasons. However, non-reducing sugars were the most important carbohydrate that apparently regulated photosynthesis on a typical summer day, whereas starch was important on a typical winter day. As a novelty, photosynthesis stimulation by carbohydrate consumption was approximately three times higher during the summer, i.e. the growing season. Under subtropical conditions, citrus leaves exhibited relatively high photosynthesis and high carbohydrate levels on the summer day, as well as a high nocturnal consumption of starch and soluble sugars. A positive association was determined between photosynthesis and photoassimilate consumption/exportation, even in leaves showing a high carbohydrate concentration. This paper provides evidence that photosynthesis in citrus leaves is regulated by an increase in sink demand rather than by the absolute carbohydrate concentration in leaves.

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

Myelography in orange-spined-hairy-dwarf-porcupine (Sphiggurus villosus)

The myelography procedure is reported in an adult of the Sphiggurus villosus (hairy-tree-porcupine) species coming from the wild, in the region of Curitiba/PR. After proceeding with the examination, while the animal was under general anesthesia, there was a dorsal deviation of the contrast in the height of the seventh thoracic vertebra (T-7) caused by compression in the ventral region of the vertebra. This is suggestive of edema caused by intramedullary hemorrhage secondary to trauma suffered by the animal in the wild. Therefore, the use of myelography in Sphiggurus villosus (hairy-tree-porcupine) proved to be quite valid and efficient, presenting the advantages of greater precision to delimit and locate the lesion present in the vertebral column.

Broad combined orange-red emissions from Eu2+- and Eu3+-doped low-silica calcium aluminosilicate glass

In this paper, a broad combined orange-red emission from Eu2+- and Eu3+-doped low-silica calcium aluminosilicate (LSCAS) glass is reported. Spectroscopic results demonstrate that it is possible to tune the emission wavelength by changing the excitation wavelength in the UV-Vis region. The color coordinates for the emission spectra were calculated, and using the Commission Internationale de l'Eclairage 1931 and 1976 chromatic diagrams, it is possible to note that they are dependent on the excitation wavelength. In addition, the (u', v') color coordinates for the investigated LSCAS samples are close to the Planckian spectrum in the cold region between 2000 and 2600K. Our results show that the Eu:LSCAS system can be used in a white light phosphor when mixed in aggregate with phosphors using green-yellow luminescent ions. (c) 2012 Optical Society of America

Antioxidant capacity and mineral content of pulp and peel from commercial cultivars of citrus from Brazil

Four Citrus species (C. sinensis, cvs. Pera and Lima; C. latifolia Tanaka cv. Tahiti; C limettioides Tanaka cv. Sweet lime and C. reticulate, cv. Ponkan) grown in Brazil were characterised in relation to contents of minerals, ascorbic acid, total polyphenols and antioxidant capacity of pulps and peels. In general, the peels demonstrated significantly higher contents of all compounds than the pulps (p < 0.05), with the exception of the Pera orange pulp that presented the highest acid ascorbic content (68 mg/100 ml), while the Tahiti lime peel presented the lowest (8 mg/100 g). Citrus showed high levels of potassium, calcium and magnesium, and the peels were considered sources of these minerals. The Ponkan mandarin peel presented the highest antioxidant capacity. The antioxidant capacity of citrus was correlated both to vitamin C and phenolics. Aside from citrus pulps, the peels are also good sources of bioactive compounds and minerals, and can be explored for their health promoting values in food products. (C) 2012 Elsevier Ltd. All rights reserved.

Natural parasitism of Diaphorina citri Kuwayama (Hemiptera, Psyllidae) nymphs by Tamarixia radiata Waterston (Hymenoptera, Eulophidae) in Sao Paulo orange groves

Natural parasitism of Diaphorina citri Kuwayama (Hemiptera, Psyllidae) nymphs by Tamarixia radiata Waterston (Hymenoptera, Eulophidae) in Sao Paulo orange groves. The psyllid Diaphorina citri Kuwayama 1908 has become the main citrus pest species in the state of Sao Paulo, Brazil, after the introduction of the huanglongbing or citrus greening. This study evaluated the parasitism of 3rd, 4th and 5th instar D. citri nymphs by Tamarixia radiata (Waterston, 1922) in citrus groves under a regimen of regular insecticide applications in ten producing regions: Araraquara, Barretos, Bauru, Botucatu, Franca, Itapetininga, Jau, Limeira, Lins and Sao Joao da Boa Vista. Sixty-nine samples of new branches infested with nymphs of D. citri were collected from 2005 to 2008 in orange groves ranging from 1 to 20 years old, of the varieties Hamlin, Pera, Valencia and Natal. The parasitoid T. radiata is widely distributed in Sao Paulo orange groves, and was identified in 50 (72%) of the samples, showing a mean parasitism rate of 12.4%. The highest parasitism rate was observed in the "summer" (from January through March), with a mean of 25.7%. Nymphal parasitism was above 90% in two samples. The probable causes of the variations in parasitism of D. citri by T. radiata are discussed.

The Role of Molecular Conformation and Polarizable Embedding for One- and Two-Photon Absorption of Disperse Orange 3 in Solution

Solvent effects on the one- and two-photon absorption (IPA and 2PA) of disperse orange 3 (DO3) in dimethyl sulfoxide (DMSO) are studied using a discrete polarizable embedding (PE) response theory. The scheme comprises a quantum region containing the chromophore and an atomically granulated classical region for the solvent accounting for full interactions within and between the two regions. Either classical molecular dynamics (MD) or hybrid Car-Parrinello (CP) quantum/classical (QM/MM) molecular dynamics simulations are employed to describe the solvation of DO3 in DMSO, allowing for an analysis of the effect of the intermolecular short-range repulsion, long-range attraction, and electrostatic interactions on the conformational changes of the chromophore and also the effect of the solute-solvent polarization. PE linear response calculations are performed to verify the character, solvatochromic shift, and overlap of the two lowest energy transitions responsible for the linear absorption spectrum of DO3 in DMSO in the visible spectral region. Results of the PE linear and quadratic response calculations, performed using uncorrelated solute-solvent configurations sampled from either the classical or hybrid CP QM/MM MD simulations, are used to estimate the width of the line shape function of the two electronic lowest energy excited states, which allow a prediction of the 2PA cross-sections without the use of empirical parameters. Appropriate exchange-correlation functionals have been employed in order to describe the charge-transfer process following the electronic transitions of the chromophore in solution.

HEPATOTOXICITY ASSESSMENT OF THE AZO DYES DISPERSE ORANGE 1 (DO1), DISPERSE RED 1 (DR1,) AND DISPERSE RED 13 (DR13) IN HEPG2 CELLS

During the dyeing process in baths approximately 10 to 15% of the dyes used are lost and reach industrial effluents, thus polluting the environment. Studies showed that some classes of dyes, mainly azo dyes and their by-products, exert adverse effects on humans and local biota, since the wastewater treatment systems and water treatment plants were found to be ineffective in removing the color and reducing toxicity of some dyes. In the present study, the toxicity of the azo dyes disperse orange 1 (DO1), disperse red 1 (DR1), and disperse red 13 (DR13) was evaluated in HepG2 cells grown in monolayers or in three dimensional (3D) culture. Hepatotoxicity of the dyes was measured using 3-(4,5-dimethylthiazol-2yl)2,5-diphenyltetrazolium (MTT) and cell counting kit 8 (CCK-8) assays after 24, 48, and 72 h of incubation of cells with 3 different concentrations of the azo dyes. The dye DO1 only reduced the mitochondrial activity in HepG2 cells grown in a monolayer after 72 h incubation, while the dye DR1 showed this deleterious effect in both monolayer and 3D culture. In contrast, dye DR13 decreased the mitochondrial activity after 24, 48, and 72 h of exposure in both monolayer and 3D culture. With respect to dehydrogenase activity, only the dye DR13 diminished the activity of this enzyme after 72 h of exposure in both monolayer and 3D culture. Our results clearly demonstrated that exposure to the studied dyes induced cytotoxicity in HepG2 cells.

Inactivation of Alicyclobacillus acidoterrestris in orange juice by saponin extracts combined with heat-treatment

Alicyclobacillus acidoterrestris is a spoilage-causing bacterium in fruit juices. The inactivation of this bacterium by commercial saponin and saponin purified extract from Sapindus saponaria fruits combined with heat-treatment is described. We investigated heat treatment (87, 90, 95, and 99 degrees C) with incubation time ranging from 0 to 50 min, in both concentrated and reconstituted juice. juices were inoculated with 1.0 x 10(4) CFU/mL of A. acidoterrestris spores for the evaluation of the best temperature for inactivation. For the temperatures of 87, 90, and 95 degrees C counts of cell viability decreased rapidly within the first 10 to 20 min of incubation in both concentrated and reconstituted juices; inactivation at 99 degrees C ensued within 1 and 2 min. Combination of commercial saponin (100 mg/L) with a very short incubation time (1 min) at 99 degrees C showed a reduction of 234 log cycle for concentrated juice A. acidoterrestris spores (1.0 x 10(4) CFU/mL) in the first 24 h of incubation after treatments. The most efficient treatment was reached with 300, 400 or 500 mg/L of purified extract of saponins from S. saponaria after 5 days of incubation in concentrated juice, and after 5 days with 300 and 400 mg/L or 72 h with 500 mg/L in reconstituted juice. Commercial saponin and purified extracts from S. saponaria had similar inactivation power on A. acidoterrestris spores, without significant differences (P>0.05). Therefore, purified extract of saponins can be an alternative for the control of A acidoterrestris in fruit juices. (C) 2012 Elsevier B.V. All rights reserved.

Natural parasitism of Diaphorina citri Kuwayama (Hemiptera, Psyllidae) nymphs by Tamarixia radiata Waterston (Hymenoptera, Eulophidae) in São Paulo orange groves

The psyllid Diaphorina citri Kuwayama 1908 has become the main citrus pest species in the state of São Paulo, Brazil, after the introduction of the huanglongbing or citrus greening. This study evaluated the parasitism of 3rd, 4th and 5th instar D. citri nymphs by Tamarixia radiata (Waterston, 1922) in citrus groves under a regimen of regular insecticide applications in ten producing regions: Araraquara, Barretos, Bauru, Botucatu, Franca, Itapetininga, Jaú, Limeira, Lins and São João da Boa Vista. Sixty-nine samples of new branches infested with nymphs of D. citri were collected from 2005 to 2008 in orange groves ranging from 1 to 20 years old, of the varieties Hamlin, Pera, Valencia and Natal. The parasitoid T. radiata is widely distributed in São Paulo orange groves, and was identified in 50 (72%) of the samples, showing a mean parasitism rate of 12.4%. The highest parasitism rate was observed in the "summer" (from January through March), with a mean of 25.7%. Nymphal parasitism was above 90% in two samples. The probable causes of the variations in parasitism of D. citri by T. radiata are discussed.

Yield mapping, soil fertility and tree gaps in an orange orchard

The current high competition on Citrus industry demands from growers new management technologies for superior efficiency and sustainability. In this context, precision agriculture (PA) has developed techniques based on yield mapping and management systems that recognize field spatial variability, which contribute to increase profitability of commercial crops. Because spatial variability is often not perceived the orange orchards are still managed as uniform and adoption of PA technology on citrus farms is low. Thus, the objective of the present study was to characterize the spatial variability of three factors: fruit yield, soil fertility and occurrence of plant gaps caused by either citrus blight or huanglongbing (HLB) in a commercial Valencia orchard in Brotas, São Paulo State, Brazil. Data from volume, geographic coordinates and representative area of the bags used on harvest were recorded to generate yield points that were then interpolated to produce the yield map. Soil chemical characteristics were studied by analyzing samples collected along planting rows and inter-rows in 24 points distributed in the field. A map of density of tree gaps was produced by georeferencing individual gaps and later by counting the number of gaps within 500 m² cells. Data were submitted to statistical and geostatistical analyses. A t test was used to compare means of soil chemical characteristics between sampling regions. High variation on yield and density of tree gaps was observed from the maps. It was also demonstrated overlapping regions of high density of plant absence and low fruit yield. Soil fertility varied depending on the sampling region in the orchard. The spatial variability found on yield, soil fertility and on disease occurrence demonstrated the importance to adopt site specific nutrient management and disease control as tools to guarantee efficiency of fruit production.

Estimation of transpiration of the 'Valencia' orange young plant using thermal dissipation probe method

Most studies on measures of transpiration of plants, especially woody fruit, relies on methods of heat supply in the trunk. This study aimed to calibrate the Thermal Dissipation Probe Method (TDP) to estimate the transpiration, study the effects of natural thermal gradients and determine the relation between outside diameter and area of xylem in 'Valencia' orange young plants. TDP were installed in 40 orange plants of 15 months old, planted in boxes of 500 L, in a greenhouse. It was tested the correction of the natural thermal differences (DTN) for the estimation based on two unheated probes. The area of the conductive section was related to the outside diameter of the stem by means of polynomial regression. The equation for estimation of sap flow was calibrated having as standard lysimeter measures of a representative plant. The angular coefficient of the equation for estimating sap flow was adjusted by minimizing the absolute deviation between the sap flow and daily transpiration measured by lysimeter. Based on these results, it was concluded that the method of TDP, adjusting the original calibration and correction of the DTN, was effective in transpiration assessment.

X-Ray analysis to assess mechanical damage in sweet corn seeds

The X-ray test is a precise, fast and non-destructive method to detect mechanical damage in seeds. In the present study, the efficiency of X-ray analysis in identifying the extent of mechanical damage in sweet corn seeds and its relationship with germination and vigor was evaluated. Hybrid 'SWB 551' (sh2) seeds with round (R) and flat (F) shapes were classified as large (L), medium (M1, M2 and M3) and small (S), using sieves with round and oblong screens. After artificial exposure to different levels of damage (0, 1, 3, 5 and 7 impacts), seeds were X-rayed (15 kV, 5 min) and submitted to germination (25 °C/5 days) and cold (10 °C/7 days) tests. Digital images of normal and abnormal seedlings and ungerminated seeds from germination and cold tests were jointly analyzed with the seed X-ray images. Results showed that damage affecting the embryonic axis resulted in abnormal seedlings or dead seeds in the germination and cold tests. The X-ray analysis is efficient for identifying mechanical damage in sweet corn seeds, allowing damage severity to be associated with losses in germination and vigor.