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Introduction: The essential oil Mentha x villosa (MVEO) has a wide range of actions, including antibacterial, antifungal, antiprotozoal and schistosomicidal actions. The present study aimed to investigate the ultrastructural changes of MVEO on the tegument of adult Schistosoma mansoni., Materials and Methods: Different concentrations of MVEO were tested on S. mansoni adult worms in vitro. Ultrastructural changes on the tegument of these adult worms were evaluated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM)., Results: The MVEO caused the death of all worms at 500 μg mL(-1) after 24 h. After 24h of 500 μg mL(-1) MVEO treatment, bubble lesions were observed over the entire body of worms and they presented loss of tubercles in some regions of the ventral portion. In the evaluation by TEM, S. mansoni adult worms treated with MVEO, 500 μg mL(-1), presented changes in the tegument and vacuoles in the syncytial matrix region. Glycogen granules close to the muscle fibers were visible., Conclusion: The ability of MVEO to cause extensive ultrastructural damage to S. mansoni adult worms correlates with its schistosomicidal effects and confirms earlier findings with S. mansoni.

Accumulation of heavy metals in unconsolidated soils can prove toxic to proximal environments, if measures are not taken to stabilize soils. One way to minimize the toxicity of metals in soils is the use of materials capable of immobilizing these contaminants by sorption. Biochar (BC) can retain large amounts of heavy metals due to, among other characteristics, its large surface area. In the current experiment, sugarcane-straw-derived biochar, produced at 700 °C, was applied to a heavy-metal-contaminated mine soil at 1.5, 3.0, and 5.0% (w/w). Jack bean and Mucuna aterrima were grown in pots containing a mine contaminated soil and soil mixed with BC. Pore water was sampled to assess the effects of biochar on zinc solubility, while soils were analyzed by DTPA extraction to confirm available metal concentrations. The application of BC decreased the available concentrations of Cd, Pb, and Zn in the mine contaminated soil leading to a consistent reduction in the concentration of Zn in the pore water. Amendment with BC reduced plant uptake of Cd, Pb, and Zn with the jack bean uptaking higher amounts of Cd and Pb than M. aterrima. This study indicates that biochar application during mine soil remediation could reduce plant concentrations of heavy metals. Coupled with this, symptoms of heavy metal toxicity were absent only in plants growing in pots amended with biochar. The reduction in metal bioavailability and other modifications to the substrate induced by the application of biochar may be beneficial to the establishment of a green cover on top of mine soil to aid remediation and reduce risks.

Background: Passion fruit woodiness may be caused by Cowpea aphid-borne mosaic virus (CABMV) and is currently the major passion fruit disease in Brazil. To assess the virus-vector-host interactions, a newly introduced golden passion fruit plantation located in eastern region of São Paulo State, Brazil, was monitored., Methods: Dissemination of CABMV was determined analyzing golden passion fruit plants monthly for 18 months by PTA-ELISA. Seasonality and aphid fauna diversity was determined by identification of the captured species using yellow sticky, yellow water-pan and green tile traps. Population composition of the aphid species was determined using the descriptive index of occurrence, dominance and general classification and overlap of species in the R program., Results: Analyses of species grouping afforded to recognize 14 aphid species. The genus Aphis represented 55.42 % of the species captured. Aphid species formed two distinct clusters, one of which was characterized by the diversity of polyphagous species that presented high potential to spread CABMV., Conclusion: The low abundance and diversity of aphid species did not interfere negatively in the CABMV epidemiology. The genus Aphis, particularly Aphis fabae/solanella and A. gossypii, was crucial in the spread of CABMV in passion fruit orchards in the eastern State of São Paulo.

Huanglongbing (HLB) is one of the most severe diseases that affects citrus trees worldwide and is associated with the yet uncultured bacteria Candidatus Liberibacter spp. To assess the metabolomic differences between HLB-asymptomatic and -symptomatic tissues, extracts from leaf and root samples taken from a uniform 6-year-old commercial orchard of Valencia trees were subjected to nuclear magnetic resonance (NMR) and chemometrics. The results show that the symptomatic trees had higher sucrose content in their leaves and no variation in their roots. In addition, proline betaine and malate were detected in smaller amounts in the HLB-affected symptomatic leaves. The changes in metabolic processes of the plant in response to HLB are corroborated by the relationship between the bacterial levels and the metabolic profiles.

Heavy metals in soil are naturally occurring but may be enhanced by anthropogenic activities such as mining. Bio-accumulation of heavy metals in the food chain, following their uptake to plants can increase the ecotoxicological risks associated with remediation of contaminated soils using plants. In the current experiment sugar cane straw-derived biochar (BC), produced at 700 °C, was applied to a heavy metal contaminated mine soil at 1.5%, 3.0% and 5.0% (w/w). Jack bean (Canavalia ensiformis) and Mucuna aterrima were grown in pots containing soil and biochar mixtures, and control pots without biochar. Pore water was sampled from each pot to confirm the effects of biochar on metal solubility, whilst soils were analyzed by DTPA extraction to confirm available metal concentrations. Leaves were sampled for SEM analysis to detect possible morphological and anatomical changes. The application of BC decreased the available concentrations of Cd, Pb and Zn in 56, 50 and 54% respectively, in the mine contaminated soil leading to a consistent reduction in the concentration of Zn in the pore water (1st collect: 99 to 39 μg L(-1), 2nd: 97 to 57 μg L(-1) and 3rd: 71 to 12 μg L(-1)). The application of BC reduced the uptake of Cd, Pb and Zn by plants with the jack bean translocating high proportions of metals (especially Cd) to shoots. Metals were also taken up by Mucuna aterrima but translocation to shoot was more limited than for jack bean. There were no differences in the internal structures of leaves observed by scanning electron microscopy. This study indicates that biochar application during mine soil remediation reduce plant concentrations of potential toxic metals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The present study aimed to identify common bean (Phaseolus vulgaris L.) cultivars less susceptible to Caliothrips phaseoli (Hood) in different growing seasons, to evaluate whether climatic conditions influence plant resistance to C. phaseoli infestation, and to investigate the preferred plant part for insect feeding. Eighteen common bean cultivars were evaluated in the winter season, and 19 cultivars were assessed in the rainy and dry seasons, under field conditions in the municipality of Jaboticabal, state of São Paulo, Brazil. Infestation of C. phaseoli nymphs in the upper and lower parts of the beans plants was recorded at weekly intervals from 25 days after plant emergence (DAE) to 60 DAE. In the winter season, the cultivars 'IAC Galante,' 'IAC Centauro,' 'IAC Carioca Eté,' and 'IAC Formoso' had significantly lower number of thrips than the cultivar 'IAC Diplomata.' In the rainy season, the cultivars 'IAC Harmonia' and 'IPR Siriri' had the lowest thrips infestation, differing from the cultivars 'BRS Pontal' and 'IAC Una.' The bean cultivars were equally susceptible to C. phaseoli in the dry season. The results suggest that C. phaseoli nymphs prefer to infest leaves of the lower part of bean plants, like most generalist herbivorous insects. In the winter and dry seasons, the highest thrips infestation was observed at 60 DAE, while in the rainy season, it was recorded from 32 to 46 DAE. Overall, C. phaseoli infestation on bean cultivars was not influenced by either temperature, relative humidity, or rainfall., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Forest biophysical structure - the arrangement and frequency of leaves and stems - emerges from growth, mortality and space filling dynamics, and may also influence those dynamics by structuring light environments. To investigate this interaction, we developed models that could use LiDAR remote sensing to link leaf area profiles with tree size distributions, comparing models which did not (metabolic scaling theory) and did allow light to influence this link. We found that a light environment-to-structure link was necessary to accurately simulate tree size distributions and canopy structure in two contrasting Amazon forests. Partitioning leaf area profiles into size-class components, we found that demographic rates were related to variation in light absorption, with mortality increasing relative to growth in higher light, consistent with a light environment feedback to size distributions. Combining LiDAR with models linking forest structure and demography offers a high-throughput approach to advance theory and investigate climate-relevant tropical forest change., (© 2015 John Wiley & Sons Ltd/CNRS.)

The high species richness of tropical forests has long been recognized, yet there remains substantial uncertainty regarding the actual number of tropical tree species. Using a pantropical tree inventory database from closed canopy forests, consisting of 657,630 trees belonging to 11,371 species, we use a fitted value of Fisher's alpha and an approximate pantropical stem total to estimate the minimum number of tropical forest tree species to fall between ∼ 40,000 and ∼ 53,000, i.e., at the high end of previous estimates. Contrary to common assumption, the Indo-Pacific region was found to be as species-rich as the Neotropics, with both regions having a minimum of ∼ 19,000-25,000 tree species. Continental Africa is relatively depauperate with a minimum of ∼ 4,500-6,000 tree species. Very few species are shared among the African, American, and the Indo-Pacific regions. We provide a methodological framework for estimating species richness in trees that may help refine species richness estimates of tree-dependent taxa.

The lignin deposition in the stem of two sugarcane genotypes was assessed on exposure to water stress. The lignin content and the morphoanatomical characterization of the stem indicated that IACSP94-2094 plants are more lignified than those of IACSP95-5000 genotype, under normal water supply conditions, which was especially associated with higher lignin contents in the rind of mature internodes. Water deficit had negative impact on the biomass production, mostly with IACSP94-2094 plants, possibly due to stress severity or higher susceptibility of that genotype during the stem-lengthening phase. Water deficit led to significant alterations in the expression levels of lignin biosynthesis genes and led to an approximate 60% increase of lignin content in the rind of young internodes in both genotypes. It is concluded that the young rind region was more directly affected by water stress and, depending on the genotype, a higher lignin accumulation may occur in the stem, thus implying lower quality biomass for bioethanol production.

Strains of Xylella fastidiosa constitute a complex group of bacteria that develop within the xylem of many plant hosts, causing diseases of significant economic importance, such as Pierce's disease in North American grapevines and citrus variegated chlorosis in Brazil. X. fastidiosa has also been obtained from other host plants, in direct correlation with the development of diseases, as in the case of coffee leaf scorch (CLS)--a disease with potential to cause severe economic losses to the Brazilian coffee industry. This paper describes a thorough genomic characterization of coffee-infecting X. fastidiosa strains, initially performed through a microarray-based approach, which demonstrated that CLS strains could be subdivided in two phylogenetically distinct subgroups. Whole-genomic sequencing of two of these bacteria (one from each subgroup) allowed identification of ORFs and horizontally transferred elements (HTEs) that were specific to CLS-related X. fastidiosa strains. Such analyses confirmed the size and importance of HTEs as major mediators of chromosomal evolution amongst these bacteria, and allowed identification of differences in gene content, after comparisons were made with previously sequenced X. fastidiosa strains, isolated from alternative hosts. Although direct experimentation still needs to be performed to elucidate the biological consequences associated with such differences, it was interesting to verify that CLS-related bacteria display variations in genes that produce toxins, as well as surface-related factors (such as fimbrial adhesins and LPS) that have been shown to be involved with recognition of specific host factors in different pathogenic bacteria., (© 2015 The Authors.)

The essential oils from leaves and inflorescences of Lippia origanoides Kunth present aromatic and medicinal potential and have been used to treat several diseases, including melanoma. In Brazil, L. origanoides is commonly found in campo cerrado and cerrado stricto sensu, physiognomies featured mainly by the differential light conditions to which short and medium-sized plants are subjected. Our aim was to investigate the glandular trichome density and the yield and chemical composition of the essential oils in leaves and inflorescences of L. origanoides from campo cerrado and cerrado stricto sensu. For glandular density analysis, leaves and inflorescences were processed according to conventional techniques for scanning electron microscopy. The essential oils of leaves and inflorescences were obtained by hydrodistillation and identified with gas chromatography. Bracts and sepals showed the highest glandular density, followed by petals and leaves. The glandular density in the abaxial leaf surface was higher in individuals from the campo cerrado. In both populations the essential oil yield was higher in inflorescences than in leaves. The chemical composition of the essential oils varied among individuals from different areas and inside a same population. Our results demonstrated the chemical plasticity of L. origanoides suggesting the importance of monitoring its popular use.

Genetic resistance of common bean (Phaseolus vulgaris L.) against angular leaf spot (ALS), caused by the fungus Pseudocercospora griseola, is conferred by quantitative trait loci (QTL). In this study, we determined the gene content of the major QTL ALS10.1 located at the end of chromosome Pv10, and identified those that are responsive to ALS infection in resistant (CAL 143) and susceptible (IAC-UNA) genotypes. Based on the current version of the common bean reference genome, the ALS10.1 core region contains 323 genes. Gene Ontology (GO) analysis of these coding sequences revealed the presence of genes involved in signal perception and transduction, programmed cell death (PCD), and defense responses. Two putative R gene clusters were found at ALS10.1 containing evolutionary related coding sequences. Among them, the Phvul.010G025700 was consistently up-regulated in the infected IAC-UNA suggesting its contribution to plant susceptibility to the fungus. We identified six other genes that were regulated during common bean response to P. griseola; three of them might be negative regulators of immunity as they showed opposite expression patterns during resistant and susceptible reactions at the initial phase of fungal infection. Taken together, these findings suggest that common bean reaction to P. griseola involves transcriptional modulation of defense genes in the ALS10.1 locus, contributing to resistance or susceptibility depending on the plant-pathogen interaction.

Because of the continuous introduction of germplasm from abroad, some collections have a high number of accessions, making it difficult to explore the genetic variability present in a germplasm bank for conservation and breeding purposes. Therefore, the aim of this study was to quantify and analyze the structure of genetic variability among 500 common bean accessions to construct a core collection. A total of 58 SSRs were used for this purpose. The polymorphism information content (PIC) in the 180 common bean accessions selected to compose the core collection ranged from 0.17 to 0.86, and the discriminatory power (DP) ranged from 0.21 to 0.90. The 500 accessions were clustered into 15 distinct groups and the 180 accessions into four distinct groups in the Structure analysis. According to analysis of molecular variance, the most divergent accessions comprised 97.2% of the observed genetic variability present within the base collection, confirming the efficiency of the selection criterion. The 180 selected accessions will be used for association mapping in future studies and could be potentially used by breeders to direct new crosses and generate elite cultivars that meet current and future global market needs.

Asiatic citrus canker (ACC) is caused by Xanthomonas citri subsp. citri. The disease results in yield loss and renders fruit unfit for the fresh market. A 6-year study in Paraná State, Brazil, was conducted to compare the susceptibility of 186 genotypes of citrus representing sweet orange (Citrus sinensis), mandarin (C. reticulata), Mediterranean mandarin (C. deliciosa), Clementine mandarin (C. clementina), Satsuma mandarin (C. unshiu), sour orange (C. aurantium), lemon (C. limon), sweet lime (C. aurantifolia), grapefruit (C. paradisi), and four hybrids (C. reticulata × Citrus sp., C. reticulata × C. paradisi, C. reticulata × C. sinensis, and C. unshiu × C. sinensis). Sweet orange (C. sinensis) was represented by the most genotypes (n = 141). The number of lesions per leaf was assessed 18 times from 2005 to 2010 (up to 4 times per year). The data were analyzed using mixed-model analysis of fixed and random effects, which showed a total of six resistance-susceptibility groupings of species and hybrids. Based on species, the most resistant genotypes, on average, included Satsuma and lemon (mean lesions per leaf = 4.32 and 4.26, respectively), and the most susceptible genotypes were grapefruit and sweet lime, with 14.84 and 10.96 lesions per leaf, respectively. Genotypes of mandarin, sour orange, Mediterranean mandarin, and sweet orange had intermediate severity (5.48 to 9.56 lesions per leaf). The hybrids also showed a range of ACC severity but all were in the more resistant groupings (5.26 to 7.35 lesions per leaf). No genotype was immune to ACC. The most resistant genotype was 'Muscia' (C. reticulata) and the most susceptible was 'Valencia Frost' (C. sinensis) (1.86 and 14.78 lesions per leaf, respectively). Approximately one-sixth of the genotypes showed a negative relationship of mean lesions per leaf with time, suggesting increasing resistance as they aged, due to a reduction in either new flush or plant size and structure. These results of the relative susceptibility of different citrus genotypes can be used in future research and to assist in varietal selection or for breeding purposes both within Brazil and other regions where ACC is an issue.

Background: How to map quantitative trait loci (QTL) with epistasis efficiently and reliably has been a persistent problem for QTL mapping analysis. There are a number of difficulties for studying epistatic QTL. Linkage can impose a significant challenge for finding epistatic QTL reliably. If multiple QTL are in linkage and have interactions, searching for QTL can become a very delicate issue. A commonly used strategy that performs a two-dimensional genome scan to search for a pair of QTL with epistasis can suffer from low statistical power and also may lead to false identification due to complex linkage disequilibrium and interaction patterns., Results: To tackle the problem of complex interaction of multiple QTL with linkage, we developed a three-stage search strategy. In the first stage, main effect QTL are searched and mapped. In the second stage, epistatic QTL that interact significantly with other identified QTL are searched. In the third stage, new epistatic QTL are searched in pairs. This strategy is based on the consideration that most genetic variance is due to the main effects of QTL. Thus by first mapping those main-effect QTL, the statistical power for the second and third stages of analysis for mapping epistatic QTL can be maximized. The search for main effect QTL is robust and does not bias the search for epistatic QTL due to a genetic property associated with the orthogonal genetic model that the additive and additive by additive variances are independent despite of linkage. The model search criterion is empirically and dynamically evaluated by using a score-statistic based resampling procedure. We demonstrate through simulations that the method has good power and low false positive in the identification of QTL and epistasis., Conclusion: This method provides an effective and powerful solution to map multiple QTL with complex epistatic pattern. The method has been implemented in the user-friendly computer software Windows QTL Cartographer. This will greatly facilitate the application of the method for QTL mapping data analysis.

Asiatic citrus canker (ACC) is an important disease of citrus in Brazil and elsewhere in the world. Infection with the causal pathogen, Xanthomonas citri subsp. citri, can cause severe disease on the fruit. Visual estimation of severity is the usual method used to quantify ACC on diseased fruit. The objective of this research was to construct and validate standard area diagram (SAD) sets as assessment aids for raters to improve the accuracy and reliability of visual estimates of ACC on unripe (green) fruit of sweet orange. Two SAD sets were constructed. A five-diagram SAD set had five severities depicted (0.5, 2.0, 8.0, 27.0, and 40.0%) and a six-diagram SAD set had six severities depicted (0.5, 1.0, 3.0, 9.0, 20.0, and 40.0%). Fifteen raters evaluated 40 images of cankered, unripe fruit. Both the five- and six-diagram SAD sets significantly improved the accuracy and reliability of estimates. Agreement, measured by Lin's concordance correlation coefficient, was 0.220 to 0.913 when not using SADs, 0.814 to 0.955 when using five-diagram SAD sets, and 0.863 to 0.925 when using six-diagram SAD sets. The five-diagram SAD set was significantly more accurate and reliable compared with the six-diagram set. Possible reasons for this are discussed. Based on the results, the five-diagram SAD set is preferable to use. Although the SAD set was developed for sweet orange, it doubtless has applicability to other citrus, including grapefruit. These SAD sets should be useful for research endeavors where accurate and reliable estimates of the severity of ACC are required.

Brown rust (causal agent Puccinia melanocephala) is an important sugarcane disease that is responsible for large losses in yield worldwide. Despite its importance, little is known regarding the genetic diversity of this pathogen in the main Brazilian sugarcane cultivation areas. In this study, we characterized the genetic diversity of 34 P. melanocephala isolates from 4 Brazilian states using loci identified from an enriched simple sequence repeat (SSR) library. The aggressiveness of 3 isolates from major sugarcane cultivation areas was evaluated by inoculating an intermediately resistant and a susceptible cultivar. From the enriched library, 16 SSR-specific primers were developed, which produced scorable alleles. Of these, 4 loci were polymorphic and 12 were monomorphic for all isolates evaluated. The molecular characterization of the 34 isolates of P. melanocephala conducted using 16 SSR loci revealed the existence of low genetic variability among the isolates. The average estimated genetic distance was 0.12. Phenetic analysis based on Nei's genetic distance clustered the isolates into 2 major groups. Groups I and II included 18 and 14 isolates, respectively, and both groups contained isolates from all 4 geographic regions studied. Two isolates did not cluster with these groups. It was not possible to obtain clusters according to location or state of origin. Analysis of disease severity data revealed that the isolates did not show significant differences in aggressiveness between regions.

Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.

The aim of this study was to characterize Heliconia genotypes phenotypically using 26 qualitative descriptors. The evaluations were conducted in five flowering stems per clump in three replicates of 22 Heliconia genotypes. Data were subjected to multivariate analysis, the Mahalanobis dissimilarity measure was estimated, and the dendrogram was generated using the nearest neighbor method. From the values generated by the dissimilarity matrix and the clusters formed among the Heliconia genotypes studied, the phenotypic characterizations that best differentiated the genotypes were: pseudostem and wax green tone (light or dark green), leaf-wax petiole, the petiole hair, cleft margin at the base of the petiole, midrib underside shade of green, wax midrib underside, color sheet (light or dark green), unequal lamina base, torn limb, inflorescence-wax, position of inflorescence, bract leaf in apex, twisting of the rachis, and type of bloom. These results will be applied in the preparation of a catalog for Heliconia descriptors, in the selection of different genotypes with most promising characteristics for crosses, and for the characterization of new genotypes to be introduced in germplasm collections.

Premise of the Study: Nine microsatellite (simple sequence repeat [SSR]) loci were characterized for natural populations of Piper solmsianum, a potential source of bioactive secondary metabolites, and analyzed to assess the levels of genetic diversity in this species. •, Methods and Results: Based on an enriched library using the oligonucleotides (CT)8 and (GT)8, a total of 19 pairs of SSR primers were designed and nine of them were highly polymorphic after screening of 37 specimens from two populations. The number of alleles per locus ranged from one to six while the observed heterozygosity for polymorphic loci ranged from 0.000 to 0.875. •, Conclusions: The SSR regions characterized were informative, and the genetic markers will be useful to assess the genetic diversity and gene flow in populations of P. solmsianum.

The self-fertilization or selfing rate estimation using microsatellite markers and its impact on survival and selection rate were evaluated in families derived from polycrosses that involved parents that were widely used in sugarcane breeding in Brazil. These factors were evaluated under unfavorable natural conditions of flowering and crossing. After the germination test, the viable progeny were taken to the field for survival rate evaluation (4, 6, and 10 months) and phenotypic selection at plant cane. The selfing rate estimate based on microsatellite markers present in the progeny and absent in their female parent was 98.5 and 0% for the polycross families derived from IACSP95-5000 and SP89-1115, respectively. The survival and selection rates in the last 2 evaluations were higher for the SP89-1115 outcrossed family than the IACSP95-5000 selfed family. The IACSP95-5000 cultivar excelled either as pollen donor with fertilization capability or viable seed production even under unfavorable natural conditions of crossing. The environment influence (temperature and humidity) had an important role during the polycross.

Hevea brasiliensis is a native species of the Amazon Basin of South America and the primary source of natural rubber worldwide. Due to the occurrence of South American Leaf Blight disease in this area, rubber plantations have been extended to suboptimal regions. Rubber tree breeding is time-consuming and expensive, but molecular markers can serve as a tool for early evaluation, thus reducing time and costs. In this work, we constructed six different cDNA libraries with the aim of developing gene-targeted molecular markers for the rubber tree. A total of 8,263 reads were assembled, generating 5,025 unigenes that were analyzed; 912 expressed sequence tags (ESTs) represented new transcripts, and two sequences were highly up-regulated by cold stress. These unigenes were scanned for microsatellite (SSR) regions and single nucleotide polymorphisms (SNPs). In total, 169 novel EST-SSR markers were developed; 138 loci were polymorphic in the rubber tree, and 98 % presented transferability to six other Hevea species. Locus duplication was observed in H. brasiliensis and other species. Additionally, 43 SNP markers in 13 sequences that showed similarity to proteins involved in stress response, latex biosynthesis and developmental processes were characterized. cDNA libraries are a rich source of SSR and SNP markers and enable the identification of new transcripts. The new markers developed here will be a valuable resource for linkage mapping, QTL identification and other studies in the rubber tree and can also be used to evaluate the genetic variability of other Hevea species, which are valuable assets in rubber tree breeding.

The biochemical responses of the enzymatic antioxidant system of a drought-tolerant cultivar (IACSP 94-2094) and a commercial cultivar in Brazil (IACSP 95-5000) grown under two levels of soil water restriction (70% and 30% Soil Available Water Content) were investigated. IACSP 94-2094 exhibited one additional active superoxide dismutase (Cu/Zn-SOD VI) isoenzyme in comparison to IACSP 95-5000, possibly contributing to the heightened response of IACSP 94-2094 to the induced stress. The total glutathione reductase (GR) activity increased substantially in IACSP 94-2094 under conditions of severe water stress; however, the appearance of a new GR isoenzyme and the disappearance of another isoenzyme were found not to be related to the stress response because the cultivars from both treatment groups (control and water restrictions) exhibited identical changes. Catalase (CAT) activity seems to have a more direct role in H2O2 detoxification under water stress condition and the shift in isoenzymes in the tolerant cultivar might have contributed to this response, which may be dependent upon the location where the excessive H2O2 is being produced under stress. The improved performance of IACSP 94-2094 under drought stress was associated with a more efficient antioxidant system response, particularly under conditions of mild stress., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The silverleaf whitefly, Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae), is a cryptic species complex that contains some of the most damaging pests in tropical and subtropical regions. Recent studies have indicated that this complex is composed of at least 24 distinct and morphologically indistinguishable species that mainly differ in their ability to transmit phytoviruses, adapt to hosts, and induce physiological changes in certain hosts. The importance of this species has been increasing worldwide, because it serves as a phytovirus vector, particularly for geminiviruses, in economically important crops. Here, we aimed to examine the population variability of B. tabaci populations inhabiting 6 agricultural crops grown in 5 regions of Brazil and 1 region of the USA; BRrep [Brasília (DF, Brazil) - cabbage], ILsoj [Urbana (IL/USA) - soybean], BJabo [Bom Jesus da Lapa (BA, Brazil) - pumpkin], CPsoj [Campinas (SP, Brazil) - soybean], UBman [Ubatuba (SP, Brazil) - cassava], and PEmel [Petrolina (PE, Brazil) - melon]. Thirteen polymorphic loci with 50 alleles were observed, with an average of 2.37 (range: 2.00-2.91) alleles per population. The UBman and PEmel B. tabaci populations were the most differentiated, which was probably caused by insect adaptation to the host plant and the use of insecticides. A 33.87% inter-population variation was observed, indicating that microsatellites may be used to measure differentiation among these B. tabaci populations. Based on the comparison of microsatellites in the current study, only the Middle East-Asia Minor 1 population of B. tabaci was found in the six populations.

Citrus leprosis (CL) caused by Citrus leprosis virus C (CiLV-C) is present in Latin America from Mexico to Argentina, where citrus plants are grown. CiLV-C is transmitted by the tenuipalpid mite, Brevipalpus phoenicis, causing localized lesions on citrus leaves, fruit, and stems. One limitation to study of the virus-vector-host relationship in this pathosystem is the lack of a suitable assay plant. On Citrus spp. used as susceptible hosts, symptoms may take weeks or months to appear after experimental inoculation by viruliferous mites. Common bean (Phaseolus vulgaris) was found to respond with localized necrotic lesions after inoculation with viruliferous B. phoenicis in 5 days. Thus far, 113 tested common bean varieties and lines and some recent accessions of varied genetic background behaved in a similar way. Black bean 'IAC Una' was adopted as a standard test variety. When inoculated leaves were left at 28 to 30°C, the period for the lesion appearance was reduced to only 2 days. Confirmation that the lesions on common bean leaves are caused by CiLV-C were made by transmission electron microscopy, immunofluorescence, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction specific for CiLV-C. Common bean plants mite-inoculated with some other cytoplasmic-type Brevipalpus-transmitted viruses (BrTVs) (Passion fruit green spot virus, Solanum violaefolium ringspot virus, Ligustrum ringspot virus, and Hibiscus green spot virus) also responded with necrotic local lesions and may serve as test plants for these viruses. Two nuclear types of BrTV (Coffee ringspot virus and Clerodendrum chlorotic spot virus) were unable to produce symptoms on common bean.

Xanthomonas citri subsp. citri (Xcc) develops a biofilm structure both in vitro and in vivo. Despite all the progress achieved by studies regarding biofilm formation, many of its mechanisms remain poorly understood. This work focuses on the identification of new genes involved in biofilm formation and how they are related to motility, virulence and chemotaxis in Xcc. A Tn5 library of approximately 6000 Xcc (strain 306) mutants was generated and screened to search for biofilm formation defective strains. We identified 23 genes not previously associated with biofilm formation. The analysis of the 23 mutants not only revealed the involvement of new genes in biofilm formation, but also reinforced the importance of exopolysaccharide production, motility and cell surface structures in this process. This collection of biofilm-defective mutants underscores the multifactorial genetic programme underlying the establishment of biofilm in Xcc.

Background: Citrus huanglongbing (HLB) disease is caused by endogenous, phloem-restricted, Gram negative, uncultured bacteria named Candidatus Liberibacter africanus (CaLaf), Ca. L. asiaticus (CaLas), and Ca. L. americanus (CaLam), depending on the continent where the bacteria were first detected. The Asian citrus psyllid vector, Diaphorina citri, transmits CaLas and CaLam and both Liberibacter species are present in Brazil. Several studies of the transcriptional response of citrus plants manifesting HLB symptoms have been reported, but only for CaLas infection. This study evaluated the transcriptional reprogramming of a susceptible genotype of sweet orange challenged with CaLam, using a customized 385K microarray containing approximately 32,000 unigene transcripts. We analyzed global changes in gene expression of CaLam-infected leaves of sweet orange during the symptomatic stage of infection and compared the results with previously published microarray studies that used CaLas-infected plants. Twenty candidate genes were selected to validate the expression profiles in symptomatic and asymptomatic PCR-positive leaves infected with CaLas or CaLam., Results: The microarray analysis identified 633 differentially expressed genes during the symptomatic stage of CaLam infection. Among them, 418 (66%) were upregulated and 215 (34%) were down regulated. Five hundred and fourteen genes (81%) were orthologs of genes from Arabidopsis thaliana. Gene set enrichment analysis (GSEA) revealed that several of the transcripts encoded transporters associated with the endomembrane system, especially zinc transport. Among the most biologically relevant gene transcripts in GSEA were those related to signaling, metabolism and/or stimulus to hormones, genes responding to stress and pathogenesis, biosynthesis of secondary metabolites, oxidative stress and transcription factors belonging to different families. Real time PCR of 20 candidate genes validated the expression pattern of some genes in symptomatic and asymptomatic leaves infected with CaLam or CaLas., Conclusions: Many gene transcripts and biological processes are significantly altered upon CaLam infection. Some of them had been identified in response to CaLas infection, while others had not been previously reported. These data will be useful for selecting target genes for genetic engineering to control HLB.

Fluorescence in situ hybridization (FISH) is the most direct method for physically mapping DNA sequences on chromosomes. Fluorescence in situ hybridization mapping of meiotic chromosomes during the pachytene stage is an important tool in plant cytogenetics, because it provides high-resolution measurements of physical distances. Fluorescence in situ hybridization mapping of coffee pachytene chromosomes offers significant advantages compared with FISH mapping of somatic chromosomes, because pachytene chromosomes are 30 times longer and provide additional cytological markers. However, the application of this technique to pachytene chromosomes has been complicated by problems in making preparations of meiotic chromosomes and by difficulties in the application of standard FISH protocols. We have been able to overcome most of these obstacles in applying the FISH technique to the pachytene chromosomes of coffee plants. Digesting the external callose layer surrounding the pollen mother cells (PMCs) in conjunction with other procedures permitted suitable pachytene chromosomes to be obtained by increasing cell permeability, which allowed the probe sequences to enter the cells. For the first time, hybridization signals were registered on coffee pachytene chromosomes using the FISH technique with a repetitive sequence as a probe. We obtained slides on which 80 % of the PMCs had hybridization signals, resulting in FISH labelling with high efficiency. The procedure does not seem to be dependent on the genotype, because hybridization signals were detected in genetically different coffee plants. These findings enhance the possibilities for high-resolution physical mapping of coffee chromosomes.

The silverleaf whitefly (Bemisia tabaci) is actually a complex of morphologically indistinct species that vary in their capacity to transmit plant viruses and to induce physiological disorders in plants of economic importance. The worldwide impact of this whitefly has increased greatly, as it is a vector of various types of phytovirus, especially geminiviruses, in plants of economic importance. The adaptability of B. tabaci to many regions of the world has fostered the appearance of various biotypes that attack a broad spectrum of host plants. We developed microsatellite markers to study genetic variability and population structure of this whitefly in Brazil. Thirteen polymorphic microsatellite markers were isolated and characterized in 20 individuals from a natural population that were collected in soybean in Campinas (SP). The number of alleles per locus ranged from one to two, and the expected heterozygosity ranged from 0.000 to 0.505. These microsatellite markers will be useful for studies and management of B. tabaci. The low polymorphism found in these molecular markers is probably associated with homology of genes expressed in these markers.

We aimed to characterize the population genetic structure within and among five Bemisia tabaci (Gennadius) populations collected from different host plants and geographic regions by using microssatelites as a molecular marker. Each population was represented by 19 specimens. The host plants and geographic origins of these populations were described as follows: Pop 1: Squash Barreiras (BA); Pop 2: Cotton Barreiras (BA); Pop 3: Soybean Campinas (SP); Pop 4: Tomato Cruz das Almas (BA); and Pop 5: Soybean Rondonópolis (MT). Six polymorphic loci were observed, which discriminated 31 different alleles in the studied populations, with a mean number of alleles per population of 3.30 (2.67 - 4.00). Using Fisher's Exact test, it was observed that at least three populations were in Hardy-Weinberg equilibrium for most of the studied loci (six). The dendrogram (UPGMA) separated populations into groups mainly related to the geographic origin of the samples. Only population 5 differed from the others at a 0.15 distance (74.5% group consistency). The most similar populations were 1 and 2, with a 0.01 distance (65.3%). This is in agreement with their geographic origins and it was not consistent with host specificity. The results suggest considerable gene flow (7.3%) among all whitefly populations and indicate that a better understanding of the gene flow in populations of B. tabaci associated with different hosts is required for the management of this insect.

Background: Coffee is one of the world's most important crops; it is consumed worldwide and plays a significant role in the economy of producing countries. Coffea arabica and C. canephora are responsible for 70 and 30% of commercial production, respectively. C. arabica is an allotetraploid from a recent hybridization of the diploid species, C. canephora and C. eugenioides. C. arabica has lower genetic diversity and results in a higher quality beverage than C. canephora. Research initiatives have been launched to produce genomic and transcriptomic data about Coffea spp. as a strategy to improve breeding efficiency., Results: Assembling the expressed sequence tags (ESTs) of C. arabica and C. canephora produced by the Brazilian Coffee Genome Project and the Nestlé-Cornell Consortium revealed 32,007 clusters of C. arabica and 16,665 clusters of C. canephora. We detected different GC3 profiles between these species that are related to their genome structure and mating system. BLAST analysis revealed similarities between coffee and grape (Vitis vinifera) genes. Using KA/KS analysis, we identified coffee genes under purifying and positive selection. Protein domain and gene ontology analyses suggested differences between Coffea spp. data, mainly in relation to complex sugar synthases and nucleotide binding proteins. OrthoMCL was used to identify specific and prevalent coffee protein families when compared to five other plant species. Among the interesting families annotated are new cystatins, glycine-rich proteins and RALF-like peptides. Hierarchical clustering was used to independently group C. arabica and C. canephora expression clusters according to expression data extracted from EST libraries, resulting in the identification of differentially expressed genes. Based on these results, we emphasize gene annotation and discuss plant defenses, abiotic stress and cup quality-related functional categories., Conclusion: We present the first comprehensive genome-wide transcript profile study of C. arabica and C. canephora, which can be freely assessed by the scientific community at http://www.lge.ibi.unicamp.br/coffea. Our data reveal the presence of species-specific/prevalent genes in coffee that may help to explain particular characteristics of these two crops. The identification of differentially expressed transcripts offers a starting point for the correlation between gene expression profiles and Coffea spp. developmental traits, providing valuable insights for coffee breeding and biotechnology, especially concerning sugar metabolism and stress tolerance.

A wide array of molecular markers has been used to investigate the genetic diversity among common bean species. However, the best combination of markers for studying such diversity among common bean cultivars has yet to be determined. Few reports have examined the genetic diversity of the carioca bean, commercially one of the most important common beans in Brazil. In this study, we examined the usefulness of two molecular marker systems (simple sequence repeats - SSRs and amplified fragment length polymorphisms - AFLPs) for assessing the genetic diversity of carioca beans. The amount of information provided by Roger's modified genetic distance was used to analyze SSR data and Jaccards similarity coefficient was used for AFLP data. Seventy SSRs were polymorphic and 20 AFLP primer combinations produced 635 polymorphic bands. Molecular analysis showed that carioca genotypes were quite diverse. AFLPs revealed greater genetic differentiation and variation within the carioca genotypes (Gst = 98% and Fst = 0.83, respectively) than SSRs and provided better resolution for clustering the carioca genotypes. SSRs and AFLPs were both suitable for assessing the genetic diversity of Brazilian carioca genotypes since the number of markers used in each system provided a low coefficient of variation. However, fingerprint profiles were generated faster with AFLPs, making them a better choice for assessing genetic diversity in the carioca germplasm.

In this study, we used real-time quantitative PCR (RT-qPCR) to evaluate the expression of 32 genes of Xanthomonas axonopodis pv. citri related to pathogenicity and virulence that are also involved in copper detoxification. Nearly all of the genes were up-regulated, including copA and copB. Two genes homologous to members of the type II secretion system (xcsH and xcsC) and two involved in the degradation of plant cell wall components (pglA and pel) were the most expressed in response to an elevated copper concentration. The type II secretion system (xcs operon) and a few homologues of proteins putatively secreted by this system showed enhanced expression when the bacteria were exposed to a high concentration of copper sulfate. The enhanced expression of the genes of secretion II system during copper stress suggests that this pathway may have an important role in the adaptative response of X. axonopodis pv. citri to toxic compounds. These findings highlight the potential role of these genes in attenuating the toxicity of certain metals and could represent an important means of bacterial resistance against chemicals used to control diseases.

Lychnophora pinaster Mart. (Asteraceae) is a Brazilian medicinal plant, extensively employed in popular medicine as an anti-inflammatory, analgesic and healing agent. Thirteen polymorphic microsatellite markers were developed and optimized for L. pinaster from an enriched genomic library. The markers were used to analyse 37 plants from two native populations, generating an average number of 6.6 alleles per polymorphic locus. These loci are important tools for future studies of population genetics., (© 2009 Blackwell Publishing Ltd. No claim to original US government works.)

Xylella fastidiosa is a phytopathogen that causes diseases in different plant species. The development of disease symptoms is associated to the blockage of the xylem vessels caused by biofilm formation. In this study, we evaluated the sensitivity of biofilm and planktonic cells to copper, one of the most important antimicrobial agents used in agriculture. We measured the exopolysaccharides (EPS) content in biofilm and planktonic cells and used real-time reverse transcription polymerase chain reaction to evaluate the expression of the genes encoding proteins involved in cation/multidrug extrusion (acrA/B, mexE/czcA, and metI) and others associated with different copper resistance mechanisms (copB, cutA1, cutA2, and cutC) in the X. fastidiosa biofilm formed in two different media. We confirmed that biofilms are less susceptible to copper than planktonic cells. The amount of EPS seems to be directly related to the resistance and it varies according to the media where the cells are grown. The same was observed for gene expression. Nevertheless, some genes seem to have a greater importance in biofilm cells resistance to copper. Our results suggest a synergistic effect between diffusion barriers and other mechanisms associated with bacterial resistance in this phytopathogen. These mechanisms are important for a bacterium that is constantly under stress conditions in the host.

Drought is a major constraint for the production of common bean (Phaseolus vulgaris L.). To identify molecular responses to water deficit, we performed a differential display RT-PCR (DDRT) analysis using roots of bean plants grown aeroponically and submitted to dehydration. This allowed us to visualise 1200 DDRT bands, 8.7% of which showed a clear regulation by dehydration, and to clone 42 cDNAs, called PvD1 to PvD42. Among them, 20 early-dehydration-responsive cDNAs were selected by reverse northern that were induced or repressed before detectable water status changes and induction of ABA-regulated genes. Northern analysis for 16 PvD clones confirmed these early regulations and allowed us to identify four late dehydration-responsive genes. Their putative involvement in signalling, protein turn-over and translocation, chaperones as well as root growth modulations in response to water stress is discussed., (Copyright © 2006 Elsevier Ireland Ltd. All rights reserved.)

Aims: To have a PCR-based detection method for Xanthomonas axonopodis pv. citri (Xac) using primers designed in a specific region of its genome., Methods and Results: A Xac-specific region was identified inside the rpf gene cluster of strain IAPAR 306 in an analysis of its complete genomic sequence. Two primers were designed, Xac01 and Xac02, which, when used in a standard PCR assay, direct the amplification of a 581 bp fragment from DNA of strains belonging to Xac from different regions around the world including unusual American and Asian strains. This product was not observed when DNA from strains of the closely related X. a. aurantifolli and X. a. citrumelo were used as templates. Extracts prepared from 28 xanthomonads of other species, and epiphytic bacteria isolated from citrus also failed to produce products with these primers. Amplification was obtained from cells grown in vitro, from extracts of both fresh and dried citrus canker lesions and from washes of inoculated but asymptomatic leaf surfaces. In sensitivity tests, this PCR technique detected as few as 100 cells., Conclusions: Primers Xac01 and Xac02 provide specific and sensitive detection of Xac in all citrus tissues where the pathogen is found., Significance and Impact of the Study: This PCR-based diagnostic test is suitable for monitoring asymptomatic plants in areas where the bacteria is endemic, in plant quarantine and regulatory situations, and also for obtaining an accurate diagnosis in a very short time. These are important characteristics for any assay to be used for the management of citrus canker disease.

The role of caffeine as a chemical defense of coffee against the berry borer Hypothenemus hampei was investigated. No positive correlation was observed between resistance and caffeine content in experiments in which seeds from several coffee species presenting genetic variability for the alkaloid were exposed to adult insects. The same was observed in an experiment with coffee seeds that had their caffeine content doubled by imbibition with caffeine aqueous solutions. Other experiments showed that the attractiveness to insects was not related to the caffeine content of mature fruits. These results indicate that H. hampei has evolved an adaptation to handle the toxic effects of caffeine.

Coffee ringspot virus (CoRSV) (family Rhabdoviridae) is transmitted by Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae). Coffee ringspot disease was first reported in coffee plants from Brazil in 1939 (1). In August 2000, severe symptoms of concentric ringspots and "oak leaf" patterns on coffee leaves (Coffea arabica L. cv. Catuai) were observed during field inspections conducted in two areas of San Gabriel de Desamparados, Costa Rica. The disease caused premature fruit and leaf drop in the affected plants. Some areas within the ringspot lesions remained green on senescent leaves. Because CoRSV particles remain restricted to lesion areas (1), this virus has not been purified, and antiserum for virus detection is not available. Therefore, leaves with symptoms were collected and examined by transmission electron microscopy. In ultrathin sections of symptomatic leaves, arrays of rhabdovirus-like particles were associated with the nucleus as described for CoRSV (2). Healthy tissues did not contain similar arrays of bacilliform and bullet-shaped particles. Twenty mites collected from the infected plants at the same locations and time were slide-mounted and identified as B. phoenicis. High populations of this mite were also observed infesting plants of Cajanus cajan L. that were intercropped with coffee at the same location. Sweet orange trees growing in the same fields as shade for the coffee did not show symptoms of citrus leprosis, a disease caused by another Brevipalpus-transmitted virus that was recently reported in Panama (3). To our knowledge, this is the first report of a virus similar to CoRSV in Costa Rica. The spread of this virus, presumably CoRSV, could seriously affect the coffee industry throughout Central America by increasing production costs. It may be necessary to apply one or more foliar acaricides to effectively control the mite vector. References: (1) A. Bitancourt. O. Biol. 5:33, 1939. (2) C. M. Chagas et al. Phytopathol. Z. 102:100, 1981. (3) F. S. Dominguez et al. Plant Dis. 85:228, 2001.

The apical growth of about 20% of young tomato plants in observed fields near Campinas, State of São Paulo, Brazil, had yellow streaking of veins. Leaf symptoms developed into patches of yellow mosaic and the leaves became wavy. The whitefly Bemisia tabaci Genn. transmitted a pathogen from the infected tomato plants to healthy tomato and potato plants, reproducing the original symptoms in tomato. The apical leaves of infected potatoes showed yellow or green mottle that developed into leaf distortion with yellow blotches, symptoms indistinguishable from potato-deforming mosaic disease (2). DNA was extracted from these tomato and potato plants (1). Using DNA from the infected tomato plant, polymerase chain reaction (PCR) with the degenerate primer pair PAC1v1978/ PAV1c715 (1), which amplifies part of the rep gene (AC1 ORF), the common region (CR), and part of the cp gene (AV1 ORF), and with the primer pair PBC1v2039/PBV1c800, which amplifies part of BC1 ORF, CR, and part of BV1 ORF, gave virus-specific DNA fragments of the sizes expected from a whitefly-transmitted geminivirus. These were cloned and the complete nucleotide (sequences for DNA-A (pToYA, GenBank accession no. U79998) and DNA-B (pToYB, GenBank accession no. U80042) fragments obtained. Nucleotide identity between the CRs (184 nucleotides) was 90%, strongly indicating that those fragments correspond to a bipartite subgroup III geminivirus. PCR with the DNA from infected potato gave the expected size fragment for DNA-A. The partial sequence of the rep gene was 100% identical to the homologous sequence from the PCR fragment from the infected tomato. A search in the GenBank, EMBL, DDBJ, and PDB databases, using the BLAST program, found no identical geminivirus. The highest identity for the CR was 75% to tomato mottle geminivirus-Florida (ToMoV) and 74% to bean golden mosaic virus-Brazil. For the rep gene, the highest identity was 73% to tomato yellow leaf curl virus-Israel, an Old World geminivi-rus, followed by 71% to tomato golden mosaic virus-Brazil (TGMV) and ToMoV. For the cp gene, the highest identity was 86% to TGMV, followed by 83% to squash leaf curl geminivirus. Therefore, we propose the name tomato yellow vein streak geminivirus (ToYVSV) for this distinct virus (2). References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) J. A. C. Souza-Dias et al. Summa Phytopathol. 22:57, 1996.