Chemical composition and biological activities of essential oil from flowers of Psidium guajava (Myrtaceae) / Composição química e atividades biológicas do óleo essencial das flores de Psidium guajava (Myrtaceae)

Abstract Xylella fastidiosa is a plant-pathogenic bacterium that lives inside host xylem vessels, where it forms biofilm which is believed to be responsible for disrupting the passage of water and nutrients. Pectobacterium carotovorum is a Gram-negative plant-specific bacterium that causes not only soft rot in various plant hosts, but also blackleg in potato by plant cell wall degradation. Chagas disease, which is caused by Trypanosoma cruzi, has been commonly treated with nifurtimox and benzonidazole, two drugs that cause several side effects. As a result, the use of natural products for treating bacterial and neglected diseases has increased in recent years and plants have become a promising alternative to developing new medicines. Therefore, this study aimed to determine, for the first time, the chemical composition of essential oil from Psidium guajava flowers (PG-EO) and to evaluate its in vitro anti-Xylella fastidiosa, anti-Pectobacterium carotovorum, anti-Trypanosoma cruzi and cytotoxic activities. PG-EO was obtained by hydrodistillation in a Clevenger apparatus while its chemical composition was determined by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Major compounds identified in PG-EO were α-cadinol (37.8%), β-caryophyllene (12.2%), nerolidol (9.1%), α-selinene (8.8%), β-selinene (7.4%) and caryophyllene oxide (7.2%). Results showed that the PG-EO had strong trypanocidal activity against the trypomastigote forms of Trypanosoma cruzi (IC50 = 14.6 μg/mL), promising antibacterial activity against X. fastidiosa (MIC = 12.5 μg/mL) and P. carotovorum (MIC = 62.5 μg/mL), and moderate cytotoxicity against LLCMK2 adherent epithelial cells in the concentration range (CC50 = 250.5 μg/mL). In short, the PG-EO can be considered a new source of bioactive compounds for the development of pesticides and trypanocide drugs.

Resumo Xylella fastidiosa é uma bactéria patogênica que vive dentro dos vasos do xilema hospedeiro, onde forma um biofilme responsável por interromper a passagem de água e nutrientes. Pectobacterium carotovorum é uma bactéria Gram-negativa que causa não só podridão macia em várias plantas hospedeiras, mas também canela-preta na batata por degradação da parede celular da planta. A doença de Chagas, causada pelo Trypanosoma cruzi, é comumente tratada com nifurtimox e benzonidazol, duas drogas que causam vários efeitos colaterais. Como resultado, o uso de produtos naturais para o tratamento de doenças bacterianas e negligenciadas aumentou nos últimos anos e as plantas continuam sendo uma alternativa promissora para o desenvolvimento de novos medicamentos. Portanto, este estudo teve como objetivo determinar, pela primeira vez, a composição química do óleo essencial de flores de Psidium guajava (PG-EO) e avaliar suas propriedades anti-Xylella fastidiosa, anti-Pectobacterium carotovorum, anti-Trypanosoma cruzi e citotóxica in vitro. PG-EO foi obtido por hidrodestilação em um aparelho Clevenger, enquanto sua composição química foi determinada por cromatografia em fase gasosa com detecção por ionização por chama (CG-DIC) e por cromatografia em fase gasosa acoplada à espectrometria de massa (CG-EM). Os principais compostos identificados no PG-EO foram α-cadinol (37,8%), β-cariofileno (12,2%), nerolidol (9,1%), α-selineno (8,8%), β-selineno (7,4%) e óxido de cariofileno (7,2%). Os resultados mostraram que o PG-EO apresentou forte atividade tripanocida contra as formas tripomastigotas de T. cruzi (CI50 = 14,6 μg/mL), promissora atividade antibacteriana contra X. fastidiosa (MIC = 12,5 μg/mL) e P. carotovorum (MIC = 62,5 μg/mL) e citotoxicidade moderada contra células epiteliais aderentes (LLCMK2) na faixa de concentração (CC50 = 250,5 μg/mL). Em suma, o PG-EO pode ser considerado uma nova fonte de compostos bioativos para o desenvolvimento de pesticidas e drogas tripanocidas.

Chemical composition and biological activities of essential oil from flowers of Psidium guajava (Myrtaceae).

Xylella fastidiosa is a plant-pathogenic bacterium that lives inside host xylem vessels, where it forms biofilm which is believed to be responsible for disrupting the passage of water and nutrients. Pectobacterium carotovorum is a Gram-negative plant-specific bacterium that causes not only soft rot in various plant hosts, but also blackleg in potato by plant cell wall degradation. Chagas disease, which is caused by Trypanosoma cruzi, has been commonly treated with nifurtimox and benzonidazole, two drugs that cause several side effects. As a result, the use of natural products for treating bacterial and neglected diseases has increased in recent years and plants have become a promising alternative to developing new medicines. Therefore, this study aimed to determine, for the first time, the chemical composition of essential oil from Psidium guajava flowers (PG-EO) and to evaluate its in vitro anti-Xylella fastidiosa, anti-Pectobacterium carotovorum, anti-Trypanosoma cruzi and cytotoxic activities. PG-EO was obtained by hydrodistillation in a Clevenger apparatus while its chemical composition was determined by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Major compounds identified in PG-EO were α-cadinol (37.8%), ß-caryophyllene (12.2%), nerolidol (9.1%), α-selinene (8.8%), ß-selinene (7.4%) and caryophyllene oxide (7.2%). Results showed that the PG-EO had strong trypanocidal activity against the trypomastigote forms of Trypanosoma cruzi (IC50 = 14.6 µg/mL), promising antibacterial activity against X. fastidiosa (MIC = 12.5 µg/mL) and P. carotovorum (MIC = 62.5 µg/mL), and moderate cytotoxicity against LLCMK2 adherent epithelial cells in the concentration range (CC50 = 250.5 µg/mL). In short, the PG-EO can be considered a new source of bioactive compounds for the development of pesticides and trypanocide drugs.

Infection by the semi-persistently transmitted Tomato chlorosis virus alters the biology and behaviour of Bemisia tabaci on two potato clones.

Insect-borne plant viruses usually alter the interactions between host plant and insect vector in ways conducive to their transmission ('host manipulation hypothesis'). Most studies have tested this hypothesis with persistently and non-persistently transmitted viruses, while few have examined semi-persistently transmitted viruses. The crinivirus Tomato chlorosis virus (ToCV) is semi-persistently transmitted virus by whiteflies, and has been recently reported infecting potato plants in Brazil, where Bemisia tabaci Middle East Asia Minor 1 (MEAM1) is a competent vector. We investigated how ToCV infection modifies the interaction between potato plants and B. tabaci in ways that increase the likelihood of ToCV transmission, in two clones, one susceptible ('Agata') and the other moderately resistant (Bach-4) to B. tabaci. Whiteflies alighted and laid more eggs on ToCV-infected plants than mock-inoculated plants of Bach-4. When non-viruliferous whiteflies were released on ToCV-infected plants near mock-inoculated plants, adults moved more intensely towards non-infected plants than in the reverse condition for both clones. Feeding on ToCV-infected plants reduced egg-incubation period in both clones, but the egg-adult cycle was similar for whiteflies fed on ToCV-infected and mock-inoculated plants. Our results demonstrated that ToCV infection in potato plants alters B. tabaci behaviour and development in distinct ways depending on the host clone, with potential implications for ToCV spread.

Assessment of tissue development in fattening quails using the stable isotope technique.

We aimed to study the behaviour of tissue renewal in blood, pectoral muscle and keel of fattening quails during their growth and adulthood through carbon-13 turnover using the stable isotope technique. Three hundred male European quails were randomly subjected to six treatments. Diets fed to animals were initially based on corn and soybean meal and were replaced at 0, 7, 14, 21, 28 and 35 days of age with diets of rice grits and soybean meal. These diets have distinct isotopic signals and over time it changes; the tissue will incorporate dietary signal, and then, we can measure the return time for each tissue in each age. Treatments were T1 (0 to 21 days), T2 (7 to 35 days), T3 (14 to 42 days), T4 (21 to 56 days), T5 (28 to 63 days) and T6 (35 to 70 days). Carbon-13 turnover in tissues of the birds at the above-mentioned time periods was calculated using the first-order exponential function. The values of half-lives were 2.6, 3.6, 5.4, 6.3, 9.0 and 9.8 days for blood and 2.0, 1.7, 4.8, 6.7, 6.9 and 6.6 days for pectoral muscle in treatments T1, T2, T3, T4, T5 and T6 respectively. The half-lives for the keel in treatments T2, T3, T4, T5 and T6 were 2.0, 5.4, 7.7, 8.9 and 15 days respectively. Thus, half-life in sampled tissues generally increased with age of birds. Initially, the tissue half-lives are influenced by growth, and at the end of the growth cycle, the breast muscle tissue was the most metabolically active in either periods, followed by blood and the keel. All tissues showed a sigmoidal growth curve, which can be confirmed by half-life as a function of age.

Chemical composition and biological activities of essential oil from flowers of Psidium guajava (Myrtaceae)

Abstract Xylella fastidiosa is a plant-pathogenic bacterium that lives inside host xylem vessels, where it forms biofilm which is believed to be responsible for disrupting the passage of water and nutrients. Pectobacterium carotovorum is a Gram-negative plant-specific bacterium that causes not only soft rot in various plant hosts, but also blackleg in potato by plant cell wall degradation. Chagas disease, which is caused by Trypanosoma cruzi, has been commonly treated with nifurtimox and benzonidazole, two drugs that cause several side effects. As a result, the use of natural products for treating bacterial and neglected diseases has increased in recent years and plants have become a promising alternative to developing new medicines. Therefore, this study aimed to determine, for the first time, the chemical composition of essential oil from Psidium guajava flowers (PG-EO) and to evaluate its in vitro anti-Xylella fastidiosa, anti-Pectobacterium carotovorum, anti-Trypanosoma cruzi and cytotoxic activities. PG-EO was obtained by hydrodistillation in a Clevenger apparatus while its chemical composition was determined by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Major compounds identified in PG-EO were -cadinol (37.8%), -caryophyllene (12.2%), nerolidol (9.1%), -selinene (8.8%), -selinene (7.4%) and caryophyllene oxide (7.2%). Results showed that the PG-EO had strong trypanocidal activity against the trypomastigote forms of Trypanosoma cruzi (IC50 = 14.6 g/mL), promising antibacterial activity against X. fastidiosa (MIC = 12.5 g/mL) and P. carotovorum (MIC = 62.5 g/mL), and moderate cytotoxicity against LLCMK2 adherent epithelial cells in the concentration range (CC50 = 250.5 g/mL). In short, the PG-EO can be considered a new source of bioactive compounds for the development of pesticides and trypanocide drugs.

Resumo Xylella fastidiosa é uma bactéria patogênica que vive dentro dos vasos do xilema hospedeiro, onde forma um biofilme responsável por interromper a passagem de água e nutrientes. Pectobacterium carotovorum é uma bactéria Gram-negativa que causa não só podridão macia em várias plantas hospedeiras, mas também canela-preta na batata por degradação da parede celular da planta. A doença de Chagas, causada pelo Trypanosoma cruzi, é comumente tratada com nifurtimox e benzonidazol, duas drogas que causam vários efeitos colaterais. Como resultado, o uso de produtos naturais para o tratamento de doenças bacterianas e negligenciadas aumentou nos últimos anos e as plantas continuam sendo uma alternativa promissora para o desenvolvimento de novos medicamentos. Portanto, este estudo teve como objetivo determinar, pela primeira vez, a composição química do óleo essencial de flores de Psidium guajava (PG-EO) e avaliar suas propriedades anti-Xylella fastidiosa, anti-Pectobacterium carotovorum, anti-Trypanosoma cruzi e citotóxica in vitro. PG-EO foi obtido por hidrodestilação em um aparelho Clevenger, enquanto sua composição química foi determinada por cromatografia em fase gasosa com detecção por ionização por chama (CG-DIC) e por cromatografia em fase gasosa acoplada à espectrometria de massa (CG-EM). Os principais compostos identificados no PG-EO foram -cadinol (37,8%), -cariofileno (12,2%), nerolidol (9,1%), -selineno (8,8%), -selineno (7,4%) e óxido de cariofileno (7,2%). Os resultados mostraram que o PG-EO apresentou forte atividade tripanocida contra as formas tripomastigotas de T. cruzi (CI50 = 14,6 g/mL), promissora atividade antibacteriana contra X. fastidiosa (MIC = 12,5 g/mL) e P. carotovorum (MIC = 62,5 g/mL) e citotoxicidade moderada contra células epiteliais aderentes (LLCMK2) na faixa de concentração (CC50 = 250,5 g/mL). Em suma, o PG-EO pode ser considerado uma nova fonte de compostos bioativos para o desenvolvimento de pesticidas e drogas tripanocidas.

First Report of Beet necrotic yellow vein virus on Red Table Beet in Brazil.

Beet necrotic yellow vein virus (BNYVV) is an economically important pathogen of sugar beet (Beta vulgaris var. saccharifera) in several European, and Asian countries and in the United States (3). The virus is transmitted by the soil-inhabiting plasmodiophorid Polymyxa betae and causes the rhizomania disease of sugar beet. In November 2012, plants of B. vulgaris subsp. vulgaris cv. Boro (red table beet) exhibiting mainly severe characteristic root symptom of rhizomania were found in a commercial field located in the municipality of São José do Rio Pardo, State of São Paulo, Brazil. No characteristic virus-inducing foliar symptom was observed on diseased plants. The incidence of diseased plants was around 70% in the two visited crops. As the hairy root symptom is indicative of infection by BNYVV, the present study aimed to detect and identify this virus associated with the diseased plants. Preliminary leaf dip analysis by transmission electron microscopy revealed the presence of very few benyvirus-like particles. Total RNA was extracted from roots of three symptomatic plants and one asymptomatic plant according to Toth et al. (3). One-step reverse-transcription-polymerase chain reaction (RT-PCR) was performed as described by Morris et al. (2) with primers that amplify part of the coat protein gene at RNA2. The initial assumption that the hairy root symptom was associated with BNYVV infection was confirmed by the amplification of a fragment of ~500 bp from all three symptomatic samples. No amplicon was obtained from the asymptomatic control plant. Amplicons were directly sequenced, and the consensus nucleotide and deduced amino acid sequences showed 100% identity. The nucleotide sequence for one amplicon (Accession No. KM433683) was compared with other sequences deposited in GenBank. The nucleotide (468 nt) and deduced amino acid (156 aa) sequences shared 93 to 100 and 97 to 99% identity, respectively with the corresponding nucleotide and amino acid sequences for other isolates of type A of BNYVV. The virus was transmitted to three of 10 red table beet plants inoculated with contaminated soil, and infection was confirmed by nested RT-PCR, as described by Morris et al. (1), and nucleotide sequencing. This is the first report on the occurrence of BNYVV in Brazil, which certainly will affect the yield of red table beet in the producing region. Therefore, mapping of the occurrence of BNYVV in red table beet-producing areas in Brazil for containment of the spread of the virus is urgent. In the meantime, precautions should be taken to control the movement of contaminated soil and beet roots, carrots, or any vegetable grown on infested land that might introduce the virus to still virus-free regions. References: (1) J. Morris et al. J. Virol. Methods 95:163, 2001. (2) D. D. Sutic et al. Handbook of Plant Virus Diseases. CRC Press, Boca Raton, Florida, 1999. (3) I. K. Toth et al. Methods for the Detection and Quantification of Erwinia carotovora subsp. atroseptica (Pectobacterium carotovorum subsb. atrosepticum) on Potatoes: A Laboratory Manual. Scottish Crop Research Institute, Dundee, Scotland, 2002.

First Report of Catharanthus mosaic virus in Mandevilla in the United States.

Mandevilla (Apocynaceae) is an ornamental tropical vine popular for its bright and attractive flowers. During 2012 to 2013, 12 Mandevilla sp. samples from Minnesota and Florida nurseries were submitted for analysis at the University of Minnesota Plant Disease Clinic. Plants showed mosaic symptoms, leaf deformation, premature leaf senescence, and vine dieback. Filamentous virus particles with modal lengths 700 to 900 nm were observed by transmission electron microscopy (TEM) in partially purified preparations from symptomatic leaves. Partially purified virions were obtained using 30% sucrose cushion centrifuged at 109,000 gmax for 2 h at 10°C (5). No other virus particles were observed in these samples, nor were any observed in non-symptomatic samples. One sample was submitted as potted plant (Mandevilla 'Sunmandeho' Sun Parasol Giant White) and was kept under greenhouse conditions for subsequent analyses. Total RNA (Qiagen) was extracted from this sample, and Potyvirus was detected using the universal primers Poty S (5'-GGN AAY AAY AGY GGN CAR CC-3') and PV1 (5'-20(T)V-3') (1) by reverse transcription (RT)-PCR (3). The amplified product was the expected ~1.7-kb, corresponding to the partial nuclear inclusion body gene, the coat protein (CP) gene, and the 3' end untranslated region. The RT-PCR amplicon was cloned (NEB) and sequenced, and the 1,720-bp consensus sequence was deposited in GenBank (Accession No. KM243928). NCBI BLAST analysis at the nucleotide level revealed highest identity (83%) with an isolate of Catharanthus mosaic virus (CatMV) from Brazil (Accession No. DQ365928). Pairwise analysis of the predicted 256 amino acid CP revealed 91% identity with the CatMV Brazilian isolate (ABI94824) and 68% or less identity with other potyviruses. Two potyviruses are usually considered the same species if their CP amino acid sequences are greater than 80% identical (2). Serological analysis of the infected sample Mandevilla 'Sunmandeho' Sun Parasol Giant White using a CatMV specific antiserum (4) resulted in positive indirect ELISA reactions. CatMV has been previously reported in periwinkle (Catharanthus roseus) in Brazil (4). Based on the analyses by TEM, RT-PCR, nucleotide and amino acid sequence identities, and serological reactivity, we identify this virus as a U.S. Mandevilla isolate of CatMV. To our knowledge, this is the first report of Catharanthus mosaic virus both in the United States and in Mandevilla. References: (1) J. Chen et al. Arch Virol. 146:757, 2001. (2) A. Gibbs and K. Ohshima. Ann. Rev. Phytopathol. 48:205, 2010. (3) R. L. Jordan et al. Acta Hortic. 901:159, 2011. (4) S. C. Maciell et al. Sci. Agric. Piracicaba, Brazil. 68:687, 2011. (5) D. Mollov et al. Arch Virol. 158:1917, 2013.

Sociodemographic, biological and behavioural risk factors associated with incidence of dental caries in schoolchildren's first permanent molars: a 3-year follow-up study.

AIM: This was to investigate the incidence of dental caries on the occlusal surface of the first permanent molars of schoolchildren, and also the associated sociodemographic, behavioural and biological risk factors. MATERIALS AND METHODS: A three-year longitudinal study on dental caries of the occlusal surface of the first permanent molars was carried out in 224 children between 9 and 11 years of age, from two public schools in Brazil. Sociodemographic, behavioural and biological risk factors were correlated with the dental caries incidence. Data collection involved dental examination and a structured questionnaire. The dependent variable was the occurrence of carious lesion. Independent variables were (baseline): age, sex, previous dental treatment, tooth brushing frequency, fluoride history of use, monthly family income, mother's education level, caries experience, visible dental plaque, and the eruption stage of the teeth in question. RESULTS: Statistics revealed a 25.4% occurrence of dental caries on the occlusal surface of the first permanent molars. The presence of visible dental plaque and history of caries (baseline) were considered risk factors for the presence of carious lesions in the examined teeth (p<0.05). CONCLUSION: Dental caries on the occlusal surface of first permanent molars was associated with a history of dental caries and presence of dental plaque on the teeth in question in the first phase of the study.

First Report of Tomato yellow spot virus Infecting Leonurus sibiricus in Brazil.

In Brazil, serious epidemics of begomovirus diseases have been successively reported since the mid-90s, among them those caused by Tomato yellow spot virus (ToYSV) (1). In July 2009 and October 2010, high incidences (40 to 60%) of plants of the weed Leonurus sibiricus (Lamiaceae) exhibiting symptoms of yellow leaf mosaic were found near soybean (Glycine max) crops within the municipalities of Marechal Cândido Rondon and Tapejara, in the states of Paraná and Rio Grande do Sul. Leaves from 21 symptomatic and seven asymptomatic L. sibiricus plants were collected from both localities and tested for the presence of begomovirus. Total DNA was extracted from each sample using Dneasy Plant Mini Kit (Qiagen) and submitted to PCR using begomovirus universal oligonucleotides PAL1v1978/PAR1c496 (3). One fragment of approximately 1,300 bp comprising the 5'-region of the replication-associated protein (Rep) gene, the entire intergenic region (IR), and the 5'-region of the coat protein (CP) gene was amplified from all symptomatic, but not from asymptomatic samples. Amplified fragments corresponding to all isolates were directly sequenced and nucleotide sequence comparisons indicated 98 to 99% nucleotide identity among themselves, and 93 to 94% identity with the corresponding nucleotide sequences for the DNA-A of the begomovirus ToYSV (GenBank Accession No. DQ336350). To confirm these results, the full genome of ToYSV Mc-7 isolated from Marechal Cândido Rondon was cloned and completely sequenced by primer walking (Macrogen Seoul, Korea). The DNA-A of ToYSV Mc-7 (JX513952) was 2,592 nt long and shared 92 and 91% identity with isolates of ToYSV from Argentina (FJ538207) and Brazil (DQ336350), respectively. The DNA-B of ToYSV Mc-7 (JX513952) was 2,568 nt long and shared 91% identity with DNA-B of a Brazilian isolate of ToYSV (DQ336351). The ToYSV Mc-7 isolate is a new strain named Tomato yellow spot virus (Brazil:Marechal Candido Rondon 7:Leonurus:2009) [ToYSV-(BR:MCR7:Le:09)]. To demonstrate pathogenicity, virus-free adults of Bemisia tabaci biotype B were confined on symptomatic L. sibiricus plants for a 48-h acquisition period. The whiteflies were then transferred to healthy L. sibiricus, bean (Phaseolus vulgaris), soybean, and tomato (Solanum lycopersicum) plants. L. sibiricus plants showed the original symptoms on the leaves (five symptomatic plants, seven inoculated plants), whereas bean (3/7), soybean (4/10), and tomato plants (5/10) exhibited mild yellow leaf mosaic. The infection in these symptomatic plants was confirmed by PCR with oligonucleotides PAL1v1978/PAR1c496 (3) and subsequent direct nucleotide sequencing of the 5'-region of the CP gene, which confirmed the identity of the transmitted virus as ToYSV. ToYSV was first reported infecting tomato plants in Minas Gerais state, Brazil (1). Recently, ToYSV was found infecting bean and soybean plants in northwestern Argentina (2). Because L. sibiricus is a weed widely distributed throughout Brazil, and the ToYSV vector B. tabaci is also common, this weed may become a potential source of inoculun of ToYSV to bean, soybean, and tomato crops. To our knowledge, this is the first report of L. sibiricus as a natural host of ToYSV. References: (1) R. F. Calegario et al. Pesq. Agropec. Bras. 42:1335, 2007. (2) P. E. Rodríguez-Pardina et al. Ann. Appl. Biol. 158:69, 2011. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Fevillea trilobata as a Natural Host of Zucchini yellow mosaic virus in Brazil.

The antidote vines or nhandirobas (Fevillea trilobata L. [Cucurbitaceae]) are dioecious plant species native to the South American Neotropics (1). Genetic materials of these species are now being domesticated and evaluated as potential crops for seed-oil extraction aiming to produce biodiesel fuel (2). Plants of F. trilobata (Accession No. CNPH-001) were cultivated from seeds under open field conditions during the years 2008 through 2011 in Brasília-DF, Brazil. Approximately 200 plants exhibiting mosaic symptoms and severe leaf malformation (with typical bubble-like patches) were found in all fields every year. Apical mosaic was slightly more severe in female than in male plants. Electron microscopy examination of negatively stained extracts of symptomatic leaf tissue showed the presence of filamentous particles about 700 to 800 nm long. Analysis of ultra-thin sections of the same tissues revealed the presence of lamellar inclusions typical of a potyvirus infection. No aphid colonies were observed on field-grown F. trilobata plants. The virus was mechanically transmitted to healthy Cucurbita pepo cv. Caserta and Luffa cylindrica, causing systemic mosaic. Sap from these infected plants reacted in PTA-ELISA with polyclonal antiserum against Zucchini yellow mosaic virus (ZYMV), but not with antisera against Papaya ringspot virus - type W (PRSV-W), Cucumber mosaic virus (CMV), and Zucchini lethal chlorosis virus (ZLCV). Total RNA extracted from experimentally infected C. pepo was analyzed by RT-PCR using specific pairs of primers for the coat protein gene of ZYMV (3). A cDNA fragment of approximately 1,186 bp was amplified and the nucleotide sequence obtained by direct sequencing. Comparisons of the nucleotide (837 nt) and deduced amino acid (279 aa) sequences of the coat protein genomic segment (GenBank Accession No. JX502677) revealed 93 to 98% and 97 to 98% identity, respectively, with the corresponding nucleotide and amino acid sequences of a group of ZYMV isolates from distinct hosts (AY188994, AY279000, and NC_003224). The infection by ZYMV might cause fruit yield losses to F. trilobata. In addition, the infected F. trilobata crops might work as a reservoir of ZYMV providing inoculum to other cucurbit hosts since it has been managed as a semi-perennial crop. To our knowledge, this is the first report of the genus Fevillea as a natural host of ZYMV. References: (1) M. Nee et al. Syst. Bot. 34:704, 2009. (2) E. G. Shay. Biomass Bioenergy 4:227, 1993. (3) K. G. Thomson et al. J. Virol. Meth. 55:83, 1995.

Maternal racial origin and adverse pregnancy outcome: a cohort study.

OBJECTIVE: To examine the association between maternal racial origin and a wide range of adverse pregnancy outcomes after adjustment for confounding factors in obstetric history and maternal characteristics. METHODS: This was a retrospective study in women with singleton pregnancies attending their first routine hospital visit at 11 + 0 to 13 + 6 weeks of gestation. Data on maternal characteristics, and medical and obstetric history were collected and pregnancy outcomes ascertained. Regression analysis was performed to examine the association between racial origin and adverse pregnancy outcomes including pre-eclampsia (PE), gestational hypertension (GH), gestational diabetes mellitus (GDM), preterm delivery (PTD), small-for-gestational age (SGA), large-for-gestational age (LGA), miscarriage, stillbirth and elective and emergency Cesarean section (CS). RESULTS: The study population included 76 158 singleton pregnancies with a live fetus at 11 + 0 to 13 + 6 weeks. In addition to maternal characteristics and obstetric history, Afro-Caribbean racial origin was associated with increased risk for miscarriage, stillbirth, PE, GH, spontaneous PTD, GDM, SGA and CS. In women of South Asian racial origin there was increased risk for PE, GDM, SGA and CS, and East Asian race contributed to the prediction of GDM and SGA. CONCLUSION: Maternal racial origin should be combined with other maternal characteristics and obstetric history when calculating an individualized adjusted risk for adverse pregnancy outcome.

Maternal serum visfatin at 11-13 weeks' gestation in preeclampsia.

The objective of this study was to determine if the maternal serum levels of visfatin in the first trimester of pregnancy are altered in cases that develop preeclampsia (PE) and whether the levels are related to placental perfusion reflected in uterine artery pulsatility index (PI). Serum visfatin and uterine artery PI were measured at 11(+0)-13(+6) weeks in 80 cases that developed PE and 240 unaffected controls. The median visfatin and uterine artery PI multiple of the unaffected median (MoM) in the outcome groups was compared and the significance of the association between visfatin MoM and uterine artery PI MoM, birth weight percentile and gestation at delivery was determined. In the PE group, compared with controls, there was a significantly higher median visfatin MoM (1.35, interquartile range (IQR): 0.69-2.16 vs 1.00, IQR: 0.55-1.96, P=0.027) and uterine artery PI MoM (1.19, IQR: 0.95-1.44 vs 1.03, IQR: 0.83-1.22, P<0.0001). In the PE group, there was no significant association between serum visfatin MoM and uterine artery PI MoM (P=0.589), gestation at delivery (P=0.763) or birth weight percentile (P=0.646). Serum visfatin levels at 11-13 weeks are increased in women who develop PE by a mechanism unrelated to impaired placental perfusion.

Fetal fraction in maternal plasma cell-free DNA at 11-13 weeks' gestation: relation to maternal and fetal characteristics.

OBJECTIVE: To examine the possible effects of maternal and fetal characteristics on the fetal fraction in maternal plasma cell-free (cf) DNA at 11-13 weeks' gestation and estimate the proportion of pregnancies at high risk of non-invasive prenatal testing (NIPT) failure because the fetal fraction is less than 4%. METHODS: In 1949 singleton pregnancies at 11-13 weeks' gestation cf-DNA was extracted from maternal plasma. Chromosome-selective sequencing of non-polymorphic and polymorphic loci, where fetal alleles differ from maternal alleles, was used to determine the proportion of cf-DNA that was of fetal origin. Multivariable regression analysis was used to determine significant predictors of the fetal fraction among maternal and fetal characteristics. RESULTS: The fetal fraction decreased with increased maternal weight, it was lower in women of Afro-Caribbean origin than in Caucasians and increased with fetal crown-rump length, serum pregnancy-associated plasma protein-A, serum free ß-human chorionic gonadotropin, smoking and trisomy 21 karyotype. The median fetal fraction was 10.0% (interquartile range, 7.8-13.0%) and this decreased with maternal weight from 11.7% at 60 kg to 3.9% at 160 kg. The estimated proportion with fetal fraction below 4% increased with maternal weight from 0.7% at 60 kg to 7.1% at 100 kg and 51.1% at 160 kg. CONCLUSIONS: Fetal fraction in maternal plasma cf-DNA is affected by maternal and fetal characteristics.

Genetic and environmental influences on blood pressure and physical activity: a study of nuclear families from Muzambinho, Brazil

Blood pressure (BP) and physical activity (PA) levels are inversely associated. Since genetic factors account for the observed variation in each of these traits, it is possible that part of their association may be related to common genetic and/or environmental influences. Thus, this study was designed to estimate the genetic and environmental correlations of BP and PA phenotypes in nuclear families from Muzambinho, Brazil. Families including 236 offspring (6 to 24 years) and their 82 fathers and 122 mothers (24 to 65 years) were evaluated. BP was measured, and total PA (TPA) was assessed by an interview (commuting, occupational, leisure time, and school time PA). Quantitative genetic modeling was used to estimate maximal heritability (h²), and genetic and environmental correlations. Heritability was significant for all phenotypes (systolic BP: h² = 0.37 ± 0.10, P < 0.05; diastolic BP: h² = 0.39 ± 0.09, P < 0.05; TPA: h² = 0.24 ± 0.09, P < 0.05). Significant genetic (r g) and environmental (r e) correlations were detected between systolic and diastolic BP (r g = 0.67 ± 0.12 and r e = 0.48 ± 0.08, P < 0.05). Genetic correlations between BP and TPA were not significant, while a tendency to an environmental cross-trait correlation was found between diastolic BP and TPA (r e = -0.18 ± 0.09, P = 0.057). In conclusion, BP and PA are under genetic influences. Systolic and diastolic BP share common genes and environmental influences. Diastolic BP and TPA are probably under similar environmental influences.

Primary embryonic cells of Rhipicephalus microplus and Amblyomma cajennense ticks as a substrate for the development of Borrelia burgdorferi (strain G39/40).

Borrelia burgdorferi, the agent of Lyme borreliosis, is a spirochetes transmitted by ticks to humans and animals. Its cultivation in vitro in tick cells allows studies of its biology and provides methodology for future research in Brazil, and for the isolation of Borrelia spp. We examined in vitro the characteristics of embryonic cells of Rhipicephalus microplus and Amblyomma cajennense in cell culture and investigated the suitability of embryonic cells as a substrate for cultivation of B. burgdorferi. Subcultures were prepared from primary cultures of embrionary cells of R. microplus and A. cajennense maintained in Leibovitz's (L-15) complete medium at 28 ºC and 31 ºC, respectively. When a monolayer had formed, the L-15 was replaced with Barbour-Stoener-Kelly medium for experiments to infect cell cultures with B. burgdorferi. After 72 hours of cultivation, the spirochetes were counted using an inverted phase contrast microscope and dark-field illumination (400×). Survival, multiplication and the adherence of B. burgdorferi for embryonic cells of R. microplus and A. cajennense were observed. B. burgdorferi cultured with embryonic cells of R. microplus grew on average to a density (final count) of 2.4 × 10(7) spirochetes/mL, whereas in cell-free culture, an average of 2.5 × 10(7) spirochetes/mL were counted. When cultivated with A. cajennense cells, the final count of spirochetes was on average 1.7 × 10(7) spirochetes/mL, while spirochetes cultured under cell-free conditions replicated on average of 2.2 × 10(7) spirochetes/mL. Similar results were observed in the final count of Spirochetes cultivated in cells of R. microplus and A. cajennense, when compared with cell-free control. These results demonstrated that cells of R. microplus and A. cajennense have the potential to be used as growth substrate for B. burgdorferi in the study of its interaction with host cells.

Genetic and environmental influences on blood pressure and physical activity: a study of nuclear families from Muzambinho, Brazil.

Blood pressure (BP) and physical activity (PA) levels are inversely associated. Since genetic factors account for the observed variation in each of these traits, it is possible that part of their association may be related to common genetic and/or environmental influences. Thus, this study was designed to estimate the genetic and environmental correlations of BP and PA phenotypes in nuclear families from Muzambinho, Brazil. Families including 236 offspring (6 to 24 years) and their 82 fathers and 122 mothers (24 to 65 years) were evaluated. BP was measured, and total PA (TPA) was assessed by an interview (commuting, occupational, leisure time, and school time PA). Quantitative genetic modeling was used to estimate maximal heritability (h²), and genetic and environmental correlations. Heritability was significant for all phenotypes (systolic BP: h² = 0.37 ± 0.10, P < 0.05; diastolic BP: h² = 0.39 ± 0.09, P < 0.05; TPA: h² = 0.24 ± 0.09, P < 0.05). Significant genetic (r g) and environmental (r e) correlations were detected between systolic and diastolic BP (r g = 0.67 ± 0.12 and r e = 0.48 ± 0.08, P < 0.05). Genetic correlations between BP and TPA were not significant, while a tendency to an environmental cross-trait correlation was found between diastolic BP and TPA (r e = -0.18 ± 0.09, P = 0.057). In conclusion, BP and PA are under genetic influences. Systolic and diastolic BP share common genes and environmental influences. Diastolic BP and TPA are probably under similar environmental influences.

Primary embryonic cells of Rhipicephalus microplus and Amblyomma cajennense ticks as a substrate for the development of Borrelia burgdorferi (strain G39/40) / Células primárias embrionárias do carrapato Rhipicephalus microplus e Amblyomma cajennense como substrato para desenvolvimento de Borrelia burgdorferi (Cepa G39/40)

Borrelia burgdorferi, the agent of Lyme borreliosis, is a spirochetes transmitted by ticks to humans and animals. Its cultivation in vitro in tick cells allows studies of its biology and provides methodology for future research in Brazil, and for the isolation of Borrelia spp. We examined in vitro the characteristics of embryonic cells of Rhipicephalus microplus and Amblyomma cajennense in cell culture and investigated the suitability of embryonic cells as a substrate for cultivation of B. burgdorferi. Subcultures were prepared from primary cultures of embrionary cells of R. microplus and A. cajennense maintained in Leibovitz's (L-15) complete medium at 28 ºC and 31 ºC, respectively. When a monolayer had formed, the L-15 was replaced with Barbour-Stoener-Kelly medium for experiments to infect cell cultures with B. burgdorferi. After 72 hours of cultivation, the spirochetes were counted using an inverted phase contrast microscope and dark-field illumination (400×). Survival, multiplication and the adherence of B. burgdorferi for embryonic cells of R. microplus and A. cajennense were observed. B. burgdorferi cultured with embryonic cells of R. microplus grew on average to a density (final count) of 2.4 × 10(7) spirochetes/mL, whereas in cell-free culture, an average of 2.5 × 10(7) spirochetes/mL were counted. When cultivated with A. cajennense cells, the final count of spirochetes was on average 1.7 × 10(7) spirochetes/mL, while spirochetes cultured under cell-free conditions replicated on average of 2.2 × 10(7) spirochetes/mL. Similar results were observed in the final count of Spirochetes cultivated in cells of R. microplus and A. cajennense, when compared with cell-free control. These results demonstrated that cells of R. microplus and A. cajennense have the potential to be used as growth substrate for B. burgdorferi in the study of its interaction with host cells.

Borrelia burgodorferi, o agente da borreliose de Lyme, é uma espiroqueta transmitida por carrapatos aos seres humanos e animais. Seu cultivo in vitro em células de carrapato permite estudos de sua biologia e propicia metodologia para futuras pesquisas no Brasil, para o isolamento de Borrelia spp. Nós examinamos in vitro as características de células embrionárias de Rhipicephalus microplus e Amblyomma cajennense, e a viabilidade de utilização dessas células embrionárias como um substrato para cultivo de B.burgdorferi. Subculturas foram preparadas a partir de culturas primárias de células embrionárias de R. microplus e A. cajennense mantidas em meio Leibovitz's (L-15) completo, a 28 ºC e 31 ºC, respectivamente. Com a formação da monocamada, o L-15 foi substituído pelo meio Barbour-Stoener-Kelly, para o experimento de infecção com B. burgdorferi nas culturas de células. Após 72 horas de cultivo, realizou-se a contagem das espiroquetas, as quais foram avaliadas sob microscópio invertido de contraste de fase e campo escuro (400×). Verificou-se a sobrevivência, a multiplicação e a aderência de B. burgdorferi em células embrionárias de R. microplus e A. cajennense. No estudo da cultura de B. burgdorferi com células embrionárias de R. microplus, observou-se, na contagem final, média de 2,4 × 10(7) espiroquetas/mL; no cultivo livre de células, verificou-se média de 2,5 × 10(7) espiroquetas/mL. No cultivo de A. cajennense, a contagem final de espiroquetas foi, em média, 1,7 × 10(7) espiroquetas/mL, enquanto que, para as cultivadas livres de células, se verificou média de 2,2 × 10(7) espiroquetas/mL. Resultado semelhante foi observado na contagem final de espiroquetas cultivadas em células de R. microplus e A. cajennense, quando comparado com o controle livre de células. Estes resultados demonstraram que células de R. microplus e A. cajennense têm o potencial para serem utilizadas como substrato para o crescimento de B. burgdorferi no estudo da interação com as células do hospedeiro.

A distinct tymovirus infecting Cassia hoffmannseggii in Brazil.

Leaves of Cassia hoffmannseggii, a wild fabaceous species found in the Atlantic Forest, with a severe mosaic symptom were collected in Pernambuco State, Brazil. By transmission electron microscopy, two types of virus particles were found: the first was recognized as particles of a potyvirus, which was later identified as Cowpea aphid-borne mosaic virus; and the second was isometric and present in high concentration. The observation of vesicles at the periphery of chloroplasts suggested a tymovirus infection, which was confirmed by subsequent assays. A serological assay against several tymovirus antisera resulted in positive reaction of this tymo-like virus with an antiserum of Passion fruit yellow mosaic virus. By means of RT-PCR and using degenerated primers for the conserved region of RNA-dependent RNA polymerase (RdRp) gene of tymoviruses, a specific DNA fragment was amplified and sequenced. Based on this sequence, a specific forward primer was synthesized and successfully used to amplify the 3' terminal genome region, containing the partial RdRp gene and the complete coat protein (CP) sequences. The CP was 188 amino acids (aa) long, and the highest CP aa identity was observed with Kennedya yellow mosaic virus (61 %). Based on the current ICTV demarcation criterion, this isolate was considered as a distinct tymovirus and tentatively named as Cassia yellow mosaic-associated virus.

First Report of Tomato chlorosis virus in Potato in Brazil.

Potato plants (Solanum tuberosum cv. Ágata) exhibiting symptoms of leaf roll and interveinal chlorosis, especially on older leaves, were found in a commercial crop in the County of Cristalina, State of Goiás, Brazil in June 2011. The crop was severely infested by whitefly Bemisia tabaci biotype B. Four potato tubers from symptomatic plants were indexed for the presence of the following viruses: Tomato chlorosis virus (ToCV), Potato leaf roll virus (PLRV), Tomato severe rugose virus (ToSRV), and Potato virus Y (PVY). Total RNA was extracted separately from each tuber and used for reverse transcription (RT)-PCR using the HS-11/HS-12 primer pair, which amplifies a fragment of 587 bp from the highly conserved region of the heat shock protein (HSP-70) homolog gene reported for ToCV. The RT-PCR product was subsequently tested by nested-PCR for detection of ToCV with specific primers ToC-5/ToC-6 (2). Amplicons of 463 bp, amplified from total RNA separately extracted from three tubers, were purified and directly sequenced. Comparisons among the three consensus sequences of 448 bp (GenBank Accession Nos. JQ288896, JQ288897, and JQ288898) revealed respectively, 98, 100, and 100% identity with the reported sequence of a tomato isolate of ToCV from Brazil (GenBank Accession No. EU868927) (1). For ToSRV detection, total DNA was extracted from two tubers and a fragment of approximately 820 bp was amplified by PCR with specific primers (3). PLRV and PVY were indexed in two and three tubers, respectively, by double-antibody sandwich-ELISA (SASA, Edinburg, Scotland). Virus-free B. tabaci biotype B were separately transferred to potato and tomato leaves infected with ToCV for an acquisition access period of 24 h. Groups of 30 viruliferous whitefly were transferred to four, young, sprout-grown potato plants cv. Ágata (two plants per virus isolate) for 24-h inoculation access period. After 37 days of inoculation, one plant inoculated with the potato and tomato isolates of ToCV, respectively exhibited symptoms of leaf roll and interveinal chlorosis on order leaves, which were similar to that induced by PLRV. Experimental infection of potato plants with ToCV, which induced leaf roll symptoms resembling PLRV infection, was reported in the United States by Wisler et al. (4). The potato isolate of ToCV was also transmitted by B. tabaci to one of two inoculated tomato plants. The presence of ToCV in all inoculated plants was detected by nested-RT-PCR as described above. To our knowledge, this is the first report on detection of ToCV in field potato plants in the world. Considering that ToCV occurs in innumerous countries around the world, it is transmitted by a cosmopolitan insect, and it induces symptoms similar to PLRV, this finding triggers an alert to field dependent seed-potato multiplication, virus inspector, and certification system. References: (1) J. C. Barbosa et al. Plant Dis. 92:1709, 2008. (2) C. I. Dovas et al. Plant Dis. 86:1345, 2002. (3) F. R. Fernandes et al. Trop. Plant Pathol. 35:43, 2010. (4) G. C. Wisler et al. Plant Dis. 82:270, 1998.