Transgenic passionfruit expressing RNA derived from Cowpea aphid-borne mosaic virus is resistant to passionfruit woodiness disease

Sixteen transgenic yellow passionfruit (Passiflora spp.) plants (R0) were obtained which express a non-translatable transgenic RNA corresponding to the 3' region of the NIb gene and the 5' region of the CP gene, derived from the genome of a Brazilian isolate of Cowpea aphid-borne mosaic virus (CABMV). The transgenic plants were propagated by stem cuttings and challenged by sap inoculation with isolates CABMV-MG1 and CABMV-PE1. One transgenic plant (TE5-10) was resistant to the isolate CABMV-MG1, but susceptible to CABMV-PE1. The remaining transgenic plants developed systemic symptoms, equal to non-transformed plants, when inoculated with either isolate. The absence of virus in TE5-10 plants was confirmed by indirect ELISA. Transcription analysis of the transgene demonstrated that the TE5-10 plant did not accumulate transgenic mRNA, even before inoculation. After inoculation, viral RNA was only detected in plants inoculated with CABMV-PE1. These results confirm that the transgenic plant TE5-10 is resistant to isolate CABMV-MG1, and suggest that the resistance mechanism is post-transcriptional gene silencing, which is already activated in the transgenic plants before virus inoculation.

In vitro organogenesis of Passiflora alata

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO(3) incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA(3) and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.

Comportamento de maracujazeiros (Passiflora spp.) quanto à morte prematura

Na Universidade Estadual Paulista, Câmpus de Jaboticabal-SP, estudou-se o comportamento de Passifloráceas quanto à morte prematura de plantas, cultivadas em local com histórico da doença. O objetivo do trabalho foi avaliar o comportamento de diversos acessos de populações e espécies de maracujazeiros em relação a esta doença, sendo que as plantas resistentes deverão ser utilizadas como porta-enxertos de formas comerciais de maracujá-amarelo (Passiflora edulis Sims f. flavicarpa) e em programas de melhoramento genético. As espécies utilizadas foram P. edulis Sims, P. edulis Sims f. flavicarpa Degener, P. nitida H.B.K., P. cincinnata, P. giberti, P. laurifolia, P. morifolia, P. foetida e P. capsularis. em local com histórico da doença, plantaram-se mudas em número variável e em épocas distintas. A condução das plantas e os tratos culturais foram os recomendados para o maracujá-amarelo. A morte prematura das plantas ocorreu entre dois meses e dois anos da cultura no campo. P. giberti e P. nitida mostraram-se resistente à doença, independentemente do local de origem. Entre os demais acessos, não se encontraram fontes promissoras de resistência. Entretanto, novos acessos e novas espécies deverão ser estudadas na busca da resistência.

Incidência e severidade do vírus do endurecimento dos frutos em maracujazeiros enxertados em pé-franco

O objetivo deste trabalho foi avaliar a incidência e a severidade do vírus do endurecimento dos frutos em maracujazeiro-amarelo enxertado e pé-franco. O experimento foi conduzido no município de Adamantina-SP, no período de abril de 2006 a junho de 2007, adotando-se o delineamento em blocos ao acaso, com quatro tratamentos e oito repetições. Foram avaliados três porta-enxertos: Passiflora edulis, P. alata e P. gibertii, e plantas de pé-franco. Utilizou-se como copa o maracujazeiro-amarelo (Passiflora edulis Sims). Avaliaram-se a porcentagem de plantas com sintomas de virose e a severidade dos sintomas. As primeiras plantas com sintomas de virose ocorreram aos 90 dias do plantio das mudas no campo, atingindo, aos 180 dias, 100% de plantas com virose em P. alata e P. gibertii, e 97,5% em P. edulis e pé-franco.

Efeitos de sistemas de condução sobre o crescimento, produção, qualidade dos frutos e custos de instalação de maracujazeiro amarelo (Passiflora edulis Sims, f. flavicarpa Deg)

Pós-graduação em Agronomia - FEIS

Dynamic and steady: shear rheological properties of xanthan and guar gums dispersed in yellow passion fruit pulp (Passiflora edulis f. flavicarpa)

Yellow passion fruit pulp is unstable, presenting phase separation that can be avoided by the addition of hydrocolloids. For this purpose, xanthan and guar gum [0.3, 0.7 and 1.0% (w/w)] were added to yellow passion fruit pulp and the changes in the dynamic and steady - shear rheological behavior evaluated. Xanthan dispersions showed a more pronounced pseudoplasticity and the presence of yield stress, which was not observed in the guar gum dispersions. Cross model fitting to flow curves showed that the xanthan suspensions also had higher zero shear viscosity than the guar suspensions, and, for both gums, an increase in temperature led to lower values for this parameter. The gums showed different behavior as a function of temperature in the range of 5 - 35ºC. The activation energy of the apparent viscosity was dependent on the shear rate and gum concentration for guar, whereas for xanthan these values only varied with the concentration. The mechanical spectra were well described by the generalized Maxwell model and the xanthan dispersions showed a more elastic character than the guar dispersions, with higher values for the relaxation time. Xanthan was characterized as a weak gel, while guar presented a concentrated solution behavior. The simultaneous evaluation of temperature and concentration showed a stronger influence of the polysaccharide concentration on the apparent viscosity and the G' and G" moduli than the variation in temperature.

Genetic evaluation of the mating system in the blue-and-yellow macaw (Ara ararauna, Aves, Psittacidae) by DNA fingerprinting

More than 90% of birds are socially monogamous, although genetic studies indicate that many are often not sexually monogamous. In the present study, DNA fingerprinting was used to estimate the genetic relationships between nestlings belonging to the same broods to evaluate the mating system in the socially monogamous macaw, Ara ararauna. We found that in 10 of 11 broods investigated, the nestlings showed genetic similarity levels congruent with values expected among full-sibs, suggesting that they shared the same parents. However, in one brood, the low genetic similarity observed between nestlings could be a result of intraspecific brood parasitism, intraspecific nest competition or extra-pair paternity. These results, along with available behavioral and life-history data, imply that the blue-and-yellow macaw is not only socially, but also genetically monogamous. However, the occurrence of eventual cases of extra-pair paternity cannot be excluded.

Biotin-avidin sandwich elisa with specific human isotypes IgG1 and IgG4 for Culicidae mosquito blood meal identification from an epizootic yellow fever area in Brazil

With a view toward investigating the feeding behavior of Culicidae mosquitoes from an area of epizootic yellow fever transmission in the municipalities of Garruchos and Santo Antônio das Missões, Rio Grande do Sul State, Brazil, specimens were collected by aspiration from September 2005 to April 2007. The engorged females were submitted to blood meal identification by enzyme-linked immunosorbent assay (ELISA). A total of 142 blood-engorged samples were examined for human or monkey blood through species-specific IgG. Additional tests for specificity utilizing isotypes IgG1 and IgG4 of human monoclonal antibodies showed that only anti-human IgG1 was effective in recognizing blood meals of human origin. The results indicated a significant difference (p = 0.027) in detection patterns in samples of Haemagogus leucocelaenus recorded from human blood meals at Santo Antônio das Missões, which suggests some degree of exposure, since it was an area where epizootic outbreaks have been reported.

Circulation of antibodies against yellow fever virus in a simian population in the area of Porto Primavera Hydroelectric Plant, São Paulo, Brazil

Yellow fever (YF) is an acute viral infectious disease transmitted by mosquitoes which occurs in two distinct epidemiological cycles: sylvatic and urban. In the sylvatic cycle, the virus is maintained by monkey's infection and transovarian transmission in vectors. Surveillance of non-human primates is required for the detection of viral circulation during epizootics, and for the identification of unaffected or transition areas. An ELISA (enzyme-linked immunosorbent assay) was standardized for estimation of the prevalence of IgG antibodies against yellow fever virus in monkey sera (Alouatta caraya) from the reservoir area of Porto Primavera Hydroelectric Plant, in the state of São Paulo, Brazil. A total of 570 monkey sera samples were tested and none was reactive to antibodies against yellow fever virus. The results corroborate the epidemiology of yellow fever in the area. Even though it is considered a transition area, there were no reports to date of epizootics or yellow fever outbreaks in humans. Also, entomological investigations did not detect the presence of vectors of this arbovirus infection. ELISA proved to be fast, sensitive, an adequate assay, and an instrument for active search in the epidemiological surveillance of yellow fever allowing the implementation of prevention actions, even before the occurrence of epizootics.

Circulation of antibodies against yellow fever virus in a simian population in the area of Porto Primavera hydroeletric plant, São Paulo, Brazil

A febre amarela (FA) é doença infecciosa aguda de origem viral transmitida por mosquitos. No ciclo silvestre, o vírus é mantido por meio da infecção de macacos e da transmissão transovariana nos vetores. A vigilância sobre populações de primatas não humanos torna-se necessária para detectar a circulação viral, quando ainda está restrito a epizootias, e para determinar sua presença em regiões indenes ou de transição para a doença. Padronizou-se a técnica ELISA (Enzyme Linked Immunosorbent Assay) para determinar a prevalência de anticorpos da classe IgG contra o vírus da FA em soros de bugios (Alouatta caraya) da região do reservatório da Usina Hidrelétrica de Porto Primavera, SP. Foram testados soros de 570 macacos sendo que nenhuma amostra mostrou-se reativa para a presença de anticorpos contra o vírus da FA. Os resultados são coerentes com a epidemiologia da FA na região. Mesmo sendo área de transição, não se conhece, até o momento, ocorrência de epizootia ou surto de FA em humanos e investigações entomológicas não apontaram a presença de vetores para esta arbovirose. A técnica mostrou-se sensível, rápida e útil à vigilância epidemiológica como instrumento de busca ativa permitindo desencadear ações preventivas, como vacinação, antes mesmo do surgimento de epizootias

Yellow Fever Virus in Haemagogus leucocelaenus and Aedes serratus Mosquitoes, Southern Brazil, 2008

Yellow fever virus (YFV) was isolated from Haemagogus leucocelaenus mosquitoes during an epizootic in 2001 in the Rio Grande do Sul State in southern Brazil In October 2008 a yellow fever outbreak was reported there with nonhuman primate deaths and human cases This latter outbreak led to intensification of surveillance measures for early detection of YFV and support for vaccination programs We report entomologic surveillance in 2 municipalities that recorded nonhuman primate deaths Mosquitoes were collected at ground level identified and processed for virus isolation and molecular analyses Eight YFV strains were isolated (7 from pools of Hg leucocelaenus mosquitoes and another from Aedes serratus mosquitoes) 6 were sequenced and they grouped in the YFV South American genotype I The results confirmed the role of Hg leucocelaenus mosquitoes as the main YFV vector in southern Brazil and suggest that Ae serratus mosquitoes may have a potential role as a secondary vector

CONTRASTING PHYLOGEOGRAPHIC PATTERNS IN MITOCHONDRIAL DNA AND MICROSATELLITES: EVIDENCE OF FEMALE PHILOPATRY AND MALE-BIASED GENE FLOW AMONG REGIONAL POPULATIONS OF THE BLUE-AND-YELLOW MACAW (PSITTACIFORMES: ARA ARARAUNA) IN BRAZIL

Comparing the patterns of population differentiation among genetic markers with different modes of inheritance call provide insights into patterns of sex-biased dispersal and gene flow. The blue-and-yellow Macaw (Ara ararauna) is a Neotropical parrot with a broad geographic distribution ill South America. However, little is known about the natural history and current status Of remaining wild populations, including levels of genetic variability. The progressive decline and possible fragmentation of populations may endanger this species in the near future. We analyzed mitochondrial DNA (mtDNA) control-region sequences and six microsatellite 106 Of Blue-and-yellow Macaws sampled throughout their geographic range ill Brazil to describe population genetic Structure, to make inferences about historical demography and dispersal behavior, and to provide insight for conservation efforts. Analyses of population genetic structure based on mtDNA showed evidence of two major populations ill western and eastern Brazil that share a few low-frequency haplotypes. This phylogeographic pattern seems to have originated by the historical isolation of Blue-and-yellow Macaw populations similar to 374,000 years ago and has been maintained by restricted gene flow and female philopatry. By contrast, variation ill biparentally inherited microsatellites was not structured geographically, Male-biased dispersal and female philopatry best explain the different patterns observed in these two markers. Because females disperse less than males, the two regional populations with well-differentiated mtDNA haplogroups should be considered two different management units for conservation purposes. Received 4 November 2007 accepted 10 December 2008.

Molecular Identification and Phylogenetic Analysis of a Group 16SrI-B Phytoplasma Associated with Sugarcane Yellow Leaf Syndrome in Brazil

Yellow leaf syndrome was a serious problem in the beginning of the 1990s in Brazil, when yield losses were estimated to be around 50%. The disease is currently endemic, but it is considered potentially important. Previous studies have revealed only the presence of a luteovirus associated with the disease in Brazil. We report that a phytoplasma of 16SrI-B is also associated with this disease. This is the first demonstration of the presence of a group 16SrI-B phytoplasma in association with sugarcane yellow leaf in Brazil.

Occurrence of phytoplasma associated with sugarcane yellow leaf syndrome in three regions of Sao Paulo State, Brazil

Sugarcane yellow leaf syndrome caused serious damage to crops in the Sao Paulo State, Brazil, in the 1990`s. The syndrome was also reported in other countries and investigations into the etiology revealed an association between the disease and virus and/or phytoplasma. The disease is potentially important and occurs endemically in that State, and for this reason the present study was conducted in order to demonstrate the occurrence of phytoplasma in three traditional sugarcane-producing areas, in Sao Paulo State, through molecular detection From naturally infected plants. Symptomatic plants belonging to varieties SP71-6163, SP71-6180 and SP89-1115 were sampled from Piracicaba, Jau and Ribeirao Preto, and total DNA was extracted from foliar tissues. Nested PCR was conducted with primer pairs P1/ Tint-16F2n/R2, and the amplified products were analyzed by electrophoresis on agarose gels. Amplified DNA fragments of 1.2 kb evidenced the presence of phytoplasma in 36% of symptomatic plants and revealed its Occurrence in all sampled regions. The results demonstrated that phytoplasma is associated with the disease and that it is important to keep a safe inspection of nurseries and monitoring plants in the field, as well as to Select Sugarcane genotypes with a good level of resistance in breeding programs.

Antioxidant response of Nicotiana tabacum cv. Bright Yellow 2 cells to cadmium and nickel stress

Plant cell cultures are a suitable model system for investigation of the physiological mechanisms of tolerance to environmental stress. We have determined the effects of Cd (0.1 and 0.2 mM CdCl(2)) and Ni (0.075 and 0.75 mM NiCl(2)) on Nicotiana tabacum L. cv. Bright Yellow (TBY-2) cell suspension cultures over a 72-h period. Inhibition of growth, loss of cell viability and lipid peroxidation occurred, in general, only when the TBY-2 cells were grown at 0.2 mM CdCl(2) and at 0.75 mM NiCl(2). At 0.1 mM CdCl(2), a significant increase in growth was determined at the end of the experiment. Increases in the activities of all of the four enzymatic antioxidant defence systems tested, were induced by the two concentrations of Cd and Ni, but at different times during the period of metal exposure. Overall, the cellular antioxidant responses to Cd and Ni were similar and were apparently sufficient to avoid oxidative stress at the lower concentrations of Cd and Ni. The activities of glutathione reductase and glutathione S-transferase increased early but transiently, whereas the activities of catalase and guaiacol peroxidase increased in the latter half of the experimental period. Therefore it is likely that the metabolism of reduced glutathione was enhanced during the initial onset of the stress, while catalase and guaiacol-type peroxidase appeared to play a more important role in the antioxidant response once the stress became severe.