Functional and morphological analysis of isolates of phylloplane and rhizoplane endophytic bacteria interacting in different cocoa production systems in the Amazon.

Endophytic bacteria colonize different internal tissues of plants without damaging their cells. They can establish themselves in the same niche as other microorganisms and develop antagonistic activities against phytopathogens. There is little research on the functional and morphological characterization of these bacteria in production systems in the Amazon. Thus, the objective of this work was to functionally and morphologically characterize endophytic bacteria isolated from cocoa trees (Theobroma cacao L.) and evaluate their antagonistic potential against phytopathogens. A total of 197 endophytic bacteria isolates were obtained from leaves and roots of cocoa plants with different production systems and at different times of the year. The characterization of functional groups consisted of proteolytic, amylolytic and cellulolytic activity and ability to fix nitrogen and solubilize phosphate. Morphological diversity was evaluated mainly according to the following parameters: shape, color, size and elevation of the colony. Thirteen isolates of endophytic bacteria, selected by cluster analysis, were used to evaluate the antagonistic potential in paired trials against four species of phytopathogenic fungi. The largest amount of endophytic bacteria was isolated from the root (95.9%), in the dry season. The most expressive activities with regards to the enzyme index were amylolytic (71.9%), proteolytic (70.2%) and nitrogen fixing (38.6%), respectively. The similarity analysis formed two clusters with isolates CS R 2.4 and CS R 2.25 exhibiting 100% similarity. Five isolates displayed inhibitory activity against phytopathogenic fungi, most notably isolate TS R 2.19, which exhibited antagonistic activity against all fungi and mycelial growth inhibition rates between 25.7% and 50.7%. Understanding the interaction between endophytes in cocoa plants is important as a possible additional tool in biological control. Our studies are incipient and the first to be carried out in different cocoa production systems in the state of Pará, Brazil.

Assessing the diversity of whiteflies infesting cassava in Brazil.

BACKGROUND: The necessity of a competent vector for transmission is a primary ecological factor driving the host range expansion of plant arthropod-borne viruses, with vectors playing an essential role in disease emergence. Cassava begomoviruses severely constrain cassava production in Africa. Curiously, begomoviruses have never been reported in cassava in South America, the center of origin for this crop. It has been hypothesized that the absence of a competent vector in cassava is the reason why begomoviruses have not emerged in South America. METHODS: We performed a country-wide whitefly diversity study in cassava in Brazil. Adults and/or nymphs of whiteflies were collected from sixty-six cassava fields in the main agroecological zones of the country. A total of 1,385 individuals were genotyped based on mitochondrial cytochrome oxidase I sequences. RESULTS: A high species richness was observed, with five previously described species and two putative new ones. The prevalent species were Tetraleurodes acaciae and Bemisia tuberculata, representing over 75% of the analyzed individuals. Although we detected, for the first time, the presence of Bemisia tabaci Middle East-Asia Minor 1 (BtMEAM1) colonizing cassava in Brazil, it was not prevalent. The species composition varied across regions, with fields in the Northeast region showing a higher diversity. These results expand our knowledge of whitefly diversity in cassava and support the hypothesis that begomovirus epidemics have not occurred in cassava in Brazil due to the absence of competent vector populations. However, they indicate an ongoing adaptation process of BtMEAM1 to cassava, increasing the likelihood of begomovirus emergence in this crop.

Recombination and pseudorecombination driving the evolution of the begomoviruses Tomato severe rugose virus (ToSRV) and Tomato rugose mosaic virus (ToRMV): two recombinant DNA-A components sharing the same DNA-B.

BACKGROUND: Begomoviruses are dicot-infecting, whitefly-transmitted viruses with a genome comprised of one or two molecules of circular, single-stranded DNA. In Brazil, tomato-infecting begomoviruses have emerged as serious pathogens since the introduction of a new biotype of the insect vector in the mid-1990's. Tomato rugose mosaic virus (ToRMV) and Tomato severe rugose virus (ToSRV) are often found in tomato fields. The complete sequence of the DNA-B components of ToSRV and ToRMV show an identity of 98.2%. Additionally, the high nucleotide identity (96.2%) between their common regions indicates that these two viruses may share the same DNA-B. METHODS: Tomato seedlings were biolistically inoculated with ToSRV (DNA-A and DNA-B) and ToRMV (DNA-A and DNA-B) infectious clones in every possible combination of single or mixed infection. Symptom expression was evaluated for up to 35 days post-inoculation (dpi). DNA was extracted at 28 dpi and the presence of each viral genomic component was examined by rolling circle amplification (RCA) followed by digestion, as well as by quantitative, real-time PCR. Sequence comparisons, recombination and phylogenetic analyzes were performed using EMBOSS needle, RDP program and maximum likelihood inference, respectively. RESULTS: Symptoms in tomato plants inoculated with the different combinations of ToRMV and ToSRV DNA-A and DNA-B components consisted of a typical mosaic in all combinations. Pseudorecombinants were formed in all possible combinations. When two DNA-A or two DNA-B components were inoculated simultaneously, the ToRMV components were detected preferentially in relation to the ToSRV components. The combination of minor changes in both the Rep protein and the CR may be involved in the preferential replication of ToRMV components. Recombination and phylogenetic analyzes support the exchange of genetic material between ToRMV and ToSRV. CONCLUSIONS: ToRMV and ToSRV form viable pseudorecombinants in their natural host (Solanum lycopersicum) and share the same DNA-B. ToRMV DNA components are preferentially replicated over ToSRV components. These results indicate that the emergence of ToRMV involved both recombination and pseudorecombination, further highlighting the importance of these mechanisms in the emergence and adaptation of begomoviruses.

Synergism and negative interference during co-infection of tomato and Nicotiana benthamiana with two bipartite begomoviruses.

In Brazil, at least eight begomoviruses including Tomato rugose mosaic virus (ToRMV) and Tomato yellow spot virus (ToYSV) infect tomatoes. ToYSV symptoms in tomato and Nicotiana benthamiana appear earlier and are more severe compared to those of ToRMV. We investigated the role of several factors in this differential adaptation. To analyze infection kinetics, a single leaf was inoculated and subsequently detached after different periods of time. Viral DNA accumulation was quantified in plants, viral replication was analyzed in protoplasts, and tissue tropism was determined by in situ hybridization. Results indicate that ToYSV establishes a systemic infection and reaches a higher concentration earlier than ToRMV in both hosts. ToRMV negatively interferes with ToYSV during the initial stages of infection, but once systemic infection is established this interference ceases. In N. benthamiana, ToYSV invades the mesophyll, while ToRMV is phloem-restricted. During dual infection in this host, ToYSV releases ToRMV from the phloem.

Recombinant expression of Garlic virus C (GARV-C) capsid protein in insect cells and its potential for the production of specific antibodies.

Garlic cultivars in Brazil are infected by a complex of viruses and for some virus species, such as the allexivirus, purification of the virions is sometimes cumbersume. To overcome this problem, recombinant expression of viral proteins in heterologous systems is an alternative method for producing antibodies. The capsid gene from Garlic virus C (GarV-C), an Allexivirus, was inserted into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) generating the recombinant virus vSynGarV-C. The recombinant protein expression was confirmed by SDS-PAGE and western-blot of extracts from recombinant virus infected insect cells, where a protein band of approximately 32KDa was observed only in extracts from recombinant infected cells. This protein corresponded to the predicted size of the capsid protein of the GarV-C. A rabbit polyclonal antibody was raised against this protein, shown to be specific for the GarV-C protein in western-blot and dot-Elisa, however with a low titer.

Concepções subjacentes à prática pedagógica em uma pré-escola / Implicit conceptions in pedagocical practices at one preschool

Existem várias maneiras de se conceberem os objetivos da pré-escola e a forma como o professor deve conduzir as atividades. Como tais concepções se articulam na prática? Durante o ano letivo de 1996, observamos as atividades de uma turma de 22 pré-escolares (4 a 6 anos) da rede particular de ensino, registrando os procedimentos, os conteúdos e a duração de cada uma delas. Atividades estruturadas ocupam cerca de metade do tempo em todos os meses. Dentre estas, as de cópia; pintura, recorte e colagem, desenho livre e dirigido foram as que ocuparam mais tempo. Conteúdos relacionados á escrita e à sociedade foram enfatizados, em detrimento daqueles relacionados à Matemática e, especialmente, à Natureza. Inferimos destas práticas uma concepção de pré-escola cujo objetivo é preparar para a alfabetização, concebida como a transmissão de um código de transcrição. A ênfase nos aspectos gráficos, no ensino das letras e números dificulta a inclusão desses aspectos em contexto de comunicação real(AU)

Concepções subjacentes à prática pedagógica em uma pré-escola / Implicit conceptions in pedagocical practices at one preschool

Existem várias maneiras de se conceberem os objetivos da pré-escola e a forma como o professor deve conduzir as atividades. Como tais concepções se articulam na prática? Durante o ano letivo de 1996, observamos as atividades de uma turma de 22 pré-escolares (4 a 6 anos) da rede particular de ensino, registrando os procedimentos, os conteúdos e a duração de cada uma delas. Atividades estruturadas ocupam cerca de metade do tempo em todos os meses. Dentre estas, as de cópia; pintura, recorte e colagem, desenho livre e dirigido foram as que ocuparam mais tempo. Conteúdos relacionados á escrita e à sociedade foram enfatizados, em detrimento daqueles relacionados à Matemática e, especialmente, à Natureza. Inferimos destas práticas uma concepção de pré-escola cujo objetivo é preparar para a alfabetização, concebida como a transmissão de um código de transcrição. A ênfase nos aspectos gráficos, no ensino das letras e números dificulta a inclusão desses aspectos em contexto de comunicação real