Genome-Wide Analysis of Differentially Expressed Genes During the Early Stages of Tomato Infection by a Potyvirus

Plant responses against pathogens cause up-and downward shifts in gene expression. To identify differentially expressed genes in a plant-virus interaction, susceptible tomato plants were inoculated with the potyvirus Pepper yellow mosaic virus (PepYMV) and a subtractive library was constructed from inoculated leaves at 72 h after inoculation. Several genes were identified as upregulated, including genes involved in plant defense responses (e. g., pathogenesis-related protein 5), regulation of the cell cycle (e. g., cytokinin-repressed proteins), signal transduction (e. g., CAX-interacting protein 4, SNF1 kinase), transcriptional regulators (e. g., WRKY and SCARECROW transcription factors), stress response proteins (e. g., Hsp90, DNA-J, 20S proteasome alpha subunit B, translationally controlled tumor protein), ubiquitins (e. g., polyubiquitin, ubiquitin activating enzyme 2), among others. Downregulated genes were also identified, which likewise display identity with genes involved in several metabolic pathways. Differential expression of selected genes was validated by macroarray analysis and quantitative real-time polymerase chain reaction. The possible roles played by some of these genes in the viral infection cycle are discussed.

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metabolismo de glicose em gêmeos monozigóticos discordantes para aptidão cardiorrespiratória

OBJETIVO: Verificar se as concentrações de glicose e insulina em jejum são reguladas pela aptidão cardiorrespiratória (VO2máx), independentemente dos efeitos genéticos. MÉTODOS: Dados de 38 pares de gêmeos monozigóticos (11 a 18 anos) foram analisados transversalmente. Os participantes foram submetidos a um teste de esforço máximo com ergoespirometria aberta (MedGraphics VO2000® - Medical Graphics Corp., St. Paul, MN) e à coleta de sangue para estimar a concentração de glicose e insulina em jejum. A zigosidade foi determinada por intermédio da investigação de concordância dos gêmeos em relação a 15 marcadores genéticos polimórficos. Nove pares demonstraram diferença média intrapar para o consumo máximo de oxigênio ≥10mL.kg-1.min-1 e foram divididos em dois grupos, de alta e baixa aptidão. Os grupos foram comparados a partir do teste pareado de Wilcoxon, tendo em vista a assimetria dos dados. RESULTADOS: em média, os gêmeos do grupo de alta aptidão apresentaram consumo máximo de oxigênio 17% superior (13,5±3,7mL.kg-1.min-1) a seus irmãos menos aptos. Não houve diferença entre os grupos para as concentrações de insulina (36,5±34,6 versus 25,3±13,7mg/dL; p<0,813), porém, os gêmeos mais aptos demonstraram menor concentração de glicose do que seus contrapares menos aptos (82,9±7,3 versus 86,7±7,6mg/dL; p<0,010). CONCLUSÕES: Neste estudo, caracterizado como caso-controle (gêmeos monozigóticos discordantes), o irmão com menor aptidão cardiorrespiratória apresentou maior concentração de glicose em jejum, sugerindo que a baixa aptidão cardiorrespiratória está associada a distúrbios no metabolismo de glicose.

Structural and functional characteristics of selected soft wheat starches

Starches from eight soft wheat samples (two parent lines and six offspring) were isolated; relationships between their structures and properties were examined. Branch chain-length distributions of amylopectins were determined by using high-performance anion exchange chromatography equipped with an amyloglucosidase reactor and a pulsed amperometric detector (HPAEC-ENZ-PAD). Results showed that the average chain length of the eight samples varied at DP 25.6-26.9. Starch samples of lines 02, 60, 63, 95, and 114 consisted of amylopectins with more long chains (DP greater than or equal to 37) and longer average chain length (DP 26.2-26.9) than that of other samples. These starch samples of longer branch chain length displayed higher gelatinization temperatures (55.3-56.5degreesC) than that of other samples (54.4-54.9degreesC) and higher peak viscosity (110-131 RVU) and lower pasting temperature (86.3-87.6degreesC) than others (83-100 RVU and 88.2-88.9degreesC, respectively). The M-w of amylopectins, determined by using high-performance size exclusion chromatography equipped with multiangle laser-light scattering and refractive index detectors (HPSEC-MALLS-RI), were similar for all samples (6.17 x 10(8) to 6.97 x 10(8)). There were no significant differences in amylose and phosphorus contents between samples. These results indicated that physical properties of wheat starch were affected by the branch-chain length of amylopectin.

Effect of Sodium Ascorbate and the Time Lapse before Cementation after Internal Bleaching on Bond Strength between Dentin and Ceramic

Purpose: To evaluate the effects of the elapsed time (ET) after nonvital bleaching (NVB) and sodium ascorbate application (10%) (SAA) on the shear bond strength of dentin to ceramic.Materials and Methods: Bovine incisors were selected, internally bleached (35% carbamide peroxide) for 9 days and submitted to the following treatments (n = 10): G1, G2, G3-luting after 1, 7, and 14 days; G4, G5, and G6-luting after SAA, 1, 7, and 14 days, respectively. G7 and G8 were not bleached: G7-luting 24 hours after access cavity sealing; G8-luting 24 hours after access cavity sealing after SAA. After NVB, the vestibular dentin was exposed and flattened. The SAA was applied to the dentin (G4, G5, G6, G8) for 10 minutes, and it was then washed and dried. The dentin was etched (37% phosphoric acid), and an adhesive system (Single Bond 2) was applied. Feldspathic ceramic discs (VM7; 4-mm diameter, 3-mm thick) were luted with a dual-resin agent (RelyX ARC, 3M ESPE Dental Products, St. Paul, MN). After 24 hours, specimens were submitted to shear test on a universal testing machine. The data (MPa) were submitted to ANOVA and Dunnet's test (5%).Results: The means (+/- SD) obtained were (MPa): G1 (14 +/- 4.5), G2 (14.6 +/- 3.1), G3 (14 +/- 3.7), G4 (15.5 +/- 4.6), G5 (19.87 +/- 4.5), G6 (16.5 +/- 3.7), G7 (22.8 +/- 6.2), and G8 (18.9 +/- 5.4). SAA had a significant effect on bond strength (p = 0.0054). The effect of ET was not significant (p = 0.1519). G5 and G6 presented higher values than the other bleached groups (p < 0.05) and similar to G7 and G8 (p > 0.05).Conclusions: After NVB, adhesive luting to dentin is recommended after 7 days if sodium ascorbate has been applied prior to dentin hybridization.

Effect of Cement Type, Relining Procedure, and Length of Cementation on Pull-out Bond Strength of Fiber Posts

Introduction: As opposed to the cementation metal posts, the cementation of fiber posts has several details that can significantly influence the success of post retention. This study evaluated the effect of the relining procedure, the cement type, and the luted length of the post on fiber posts retention. Methods: One hundred eighty bovine incisors were selected to assess post retention; after endodontic treatment, the canals were flared with diamonds burs. Post holes were prepared in lengths of 5, 7.5, and 10 mm; the fiber posts were relined with composite resin and luted with RelyX ARC, RelyX Unicem, or RelyX Luting 2. All cements are manufactured by 3M ESPE (St. Paul, MN). Samples were subjected to a pull-out bond strength test in a universal testing machine; the results (N) were submitted to a three-way analysis of variance and the Tukey post hoc test (alpha = 0.05). Results: The improvement of post retention occurred with the increase of the post length luted into the root canal; the relining procedure improved the pull-out bond strength. RelyX Unicem and RelyX ARC showed similar values of retention, both showing higher values than RelyX Luting 2. Conclusion: Post length, the reining procedure, and the cement type are all important factors for improving the retention of fiber posts. (J Endod 2010;36:1543-1546)

The Disk-Specimen Thickness Does Not Influence the Push-Out Bond Strength Results Between Fiber Post and Root Dentin

This study evaluated the effect of different thickness of disk-shaped specimens on the push-out bond strength test. Eighteen lower bovine teeth were sectioned (20mm) and prepared (15mm) with the same post system drill (Light Post (R) #1, Schaumburg, IL, Bisco, USA). The apical third of each specimen was embedded in a plastic matrix filled with an acrylic resin (Dencrilay (TM), Dencril, São Paulo, Brazil). The posts were cleaned with alcohol, silanated (ProSil (R), FGM, Joenville, SC, Brazil) and cemented with the RelyX (TM) U100 (3M ESPE, St. Paul, MN, USA). Each specimen was sectioned into three pieces of differing thicknesses (1, 2, and 4 mm). These disk-samples were allocated into 3 groups (n=18) and subjected to push-out testing. One-way ANOVA showed no influence of the specimen thickness on the results (p=0.842). No correlation was observed between thickness and push-out bond strength (Pearson Correlation, r(2)=0.0688; P=0.6209). The push-out bond strength test was not affected by the thickness of the disk-specimens.

Pathogenic variation in Colletotrichum gloeosporioides infecting Stylosanthes spp. in a center of diversity in Brazil

Pathogenic variation in Colletotrichum gloeosporioides infecting species of the tropical pasture legume Stylosanthes at its center of diversity was determined from 296 isolates collected from wild host population and selected germ plasm of S. capitata, S. guianensis, S. scabra, and S. macrocephala in Brazil. A putative host differential set comprising 11 accessions was selected from a bioassay of 18 isolates on 19 host accessions using principal component analysis. A similar analysis of anthracnose severity data for a subset of 195 isolates on the 11 differentials indicated that an adequate summary of pathogenic variation could be obtained using only five of these differentials. of the five differentials, S. seabrana 'Primar' was resistant and S. scabra 'Fitzroy' was susceptible to most isolates. A cluster analysis was used to determine eight natural race clusters using the 195 isolates. Linear discriminant functions were developed for eight race clusters using the 195 isolates as the training data set, and these were applied to classify a test data set of the remaining 101 isolates. All except 11 isolates of the test data set were classified into one of the eight race clusters. Over 10% of the 296 isolates were weakly pathogenic to all five differentials and another 40% were virulent on just one differential. The unclassified isolates represent six new races with unique virulence combinations, of which one isolate is virulent on all five differentials. The majority of isolates came from six field sites, and Shannon's index of diversity indicated considerable variation between sites. Pathogenic diversity was extensive at three sites where selected germ plasm were under evaluation, and complex race clusters and unclassified isolates representing new races were more prevalent at these sites compared with sites containing wild Stylosanthes populations.

Efficacy of pre- and postinoculation application of Fungicides to expanding young citrus leaves for control of melanose, scab, and alternarial brown spot

In greenhouse trials, copper hydroxide, pyraclostrobin, and famoxadone were applied to actively crowing young citrus seedlings to determine the duration of protection of young leaves provided by these fungicides against melanose, caused by Diaporthe citri, citrus scab, caused by Elsinoe fawcettii, and Alternaria brown spot, caused by Alternaria alternata. Fungicides were applied to different sets of potted plants of grapefruit for control of melanose, of rough lemon for control of scab, and of Dancy tangerine for control of Afternaria brown spot 1 to 6 days prior to inoculation. as well as on the day of inoculation. Leaf area of treated shoots was estimated on the day of fungicide application and the day of inoculation and disease severity evaluated subsequently. In most cases. copper hydroxide and famoxadone provided at least 50% control of all three diseases for only about 2 days after application. Generally, there was little or no disease control when the products were applied 4 or more days before inoculation. In contrast, pyraclostrobin usually provided a high level of control of all three diseases when applied up to 5 days prior to inoculation. The level of disease control decreased as the interval between a fungicide application and inoculation increased and the relationship between disease control and leaf expansion best fit a quadratic equation. Effective disease control was observed with copper hydroxide and famoxadone until leaf area had increased by 100 to 200%, whereas control with pyraclostrobin was observed up to 400 to 500% increase in leaf area. In postinoculation tests with scab and melanose, pyraclostrobin provided high levels of disease control (>75%) when applied up to 2 days after inoculation. whereas copper hydroxide and famoxadone had minimal postinoculation activity. Applications of pyraclostrobin to the spring flush growth of citrus trees are much more likely to provide control of melanose, scab, and Alternaria brown spot than those of famoxadone or copper hydroxide.

An experimental inoculation system to study citrus-Xylella fastidiosa interactions

Difficulties in reproducing the citrus variegated chlorosis (CVC) disease symptoms in expertmental plants have delayed implementation of studies to better understand the essential aspects of this important disease. In an extensive Study, cultivars of sweet orange (Citrus sinensis) were inoculated with Xylella fastidiosa using procedures that included root immersion, and stein absorption, pricking, or infiltration of the inoculum into plants of different ages. Inoculum consisted of 5-day-old cultures or cell suspensions of CVC strain 9a5c diluted in phosphate-buffered saline. Inoculated plants and controls were grown, or transferred just after inoculation, to 5-liter pots or 72-cell foam trays. Approximately 4, 5, 9, and 12 months after inoculation, leaves were collected and processed for polymerase chain reaction analysis or X. fastidiosa isolation on BCYE agar medium. Root immersion and stem inoculation of 4- and 6-month-old plants resulted in low percentages of symptomatic (0 to 7%) and plants positive by isolation (0 to 9%). Pinpricked or injected stems of I-month-old seedlings resulted in high percentages of plants symptomatic (29 and 90% in Pera Rio, 75, 59, and 83% in Valencia, and 77% in Natal) or positive by isolation (26 and 93% in Pera Rio, 98, 96, and 83% in Valencia, and 77% in Natal), In foam trays, the seedlings grew less, the incubation period was shorter. and disease severity was higher than in pots. This system allows testing of higher numbers of plants in a reduced space with a more precise reproduction of the experimental conditions.

Effect of lesion age, humidity, and fungicide application on sporulation of Alternaria alternata, the cause of brown spot of tangerine

Alternaria brown spot, caused by Alternaria alternata, causes yield losses and fruit blemishes on many tangerines and their hybrids in most citrus areas of the world where susceptible cultivars are grown. Although the conditions affecting infection and disease severity are known, little information is available on inoculum production on infected tissue. We found that sporulation on leaves began about 10 days after symptoms developed, was abundant from 20 to 40 days, and declined thereafter. Conidial production was far greater on leaf than on fruit or twig lesions. Spore production per unit area of leaf lesion was greater on the more susceptible hybrids, Minneola and Orlando tangelos, than on the less susceptible Murcott tangor. At 74% relative humidity, conidial production on leaf lesions was low, but it was abundant at 85, 92.5, 96, and 100%. Application of Q(o)I or copper fungicides, but not ferbam, suppressed sporulation on leaf lesions for about 14 to 21 days after application. Additional applications did not appear to be more effective than a single spray in reducing inoculum production.

Evaluation of the genetic diversity of Xylella fastidiosa strains from citrus and coffee hosts by single-nucleotide polymorphism markers

The aim of this study was to obtain information about genetic diversity and make some inferences about the relationship of 27 strains of Xylella fastidiosa from different hosts and distinct geographical areas. Single-nucleotide polymorphism (SNP) molecular markers were identified in DNA sequences from 16 distinct regions of the genome of 24 strains of X. fastidiosa from coffee and citrus plants. Among the Brazilian strains, coffee-dependent strains have a greater number of SNPs (10 to 24 SNPs) than the citrus-based strains (2 to 12 SNPs); all the strains were compared with the sequenced strain 9a5c. The identified SNP markers were able to distinguish, for the first time, strains from citrus plants and coffee and showed that strains from coffee present higher genetic diversity than the others. These markers also have proven to be efficient for discriminating strains from the same host obtained from different geographic regions. X. fastidiosa, the causal agent of citrus variegated chlorosis, possesses genetic diversity, and the SNP markers were highly efficient for discriminating genetically close organisms.

An evaluation of the genetic diversity of Xylella fastidiosa isolated from diseased citrus and coffee in São Paulo, Brazil

Strains of Xylella fastidiosa, isolated from sweet orange trees (Citrus sinensis) and coffee trees (Coffea arabica) with symptoms of citrus variegated chlorosis and Requeima do Cafe, respectively, were indistinguish able based on repetitive extragenic palindromic polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus PCR assays. These strains were also indistinguishable with a previously described PCR assay that distinguished the citrus strains from all other strains of Xylella fastidiosa. Because we were not able to document any genomic diversity in our collection of Xylella fastidiosa strains isolated from diseased citrus, the observed gradient of increasing disease severity from southern to northern regions of São Paulo State is unlikely due to the presence of significantly different strains of the pathogen in the different regions. When comparisons were made to reference strains of Xylella fastidiosa isolated from other hosts using these methods, four groups were consistently identified consistent with the hosts and regions from which the strains originated: citrus and coffee, grapevine and almond, mulberry, and elm, plum, and oak. Independent results from random amplified polymorphic DNA (RAPD) PCR assays were also consistent with these results; however, two of the primers tested in RAPD-PCR were able to distinguish the coffee and citrus strains. Sequence comparisons of a PCR product amplified from all strains of Xylella fastidiosa confirmed the presence of a CfoI polymorphism that can be used to distinguish the citrus strains from all others. The ability to distinguish Xylella fastidiosa strains from citrus and coffee with a PCR-based assay will be useful in epidemiological and etiological studies of this pathogen.

Physicochemical properties of maize starch obtained from intermittent milling and dynamic steeping (IMDS) under various steeping conditions

Physicochemical properties of maize starch obtained under different steeping conditions by intermittent milling and dynamic steeping process (IMDS) were studied. Brazilian dent maize (hybrid XL 606) was milled using a 2x2x3 factorial experimental design with two lactic acid levels (0.0 and 0.55%, v/v), two SO2 levels (0.05 and 0.1%, w/v), and three temperatures (52, 60, and 68degreesC). Properties of starch obtained by conventional wet-milling process (36 hr at 52degreesC, 0.55% lactic acid, and 0.2% SO2) were used for comparison. Starch protein content and solubility increased with presence of lactic acid, while swelling power decreased. Higher SO2 concentration (0.1%) had the same effect as lactic acid on some properties. Steeping temperatures of 60 and 68degreesC increased solubility and most of the thermal properties but reduced swelling power, suggesting stronger starch annealing during IMDS at these temperatures. Some thermal changes on starch granules were visualized by scanning electron microscopy (SEM) at 60 and 68degreesC. Amylose content as well as pasting properties were affected by steeping factors and interactions. Starches from IMDS and conventional wet-milling processes were similar in most properties, indicating that IMDS provides starch with quality similar to that from conventional milling.