956 resultados para Maize bushy stunt phytoplasma


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Phytoplasmas are bacteria with a persistent propagative transmission by insect vectors that generates direct and indirect interactions among them. In order to understand these interactions for maize bushy stunt phytoplasma (MBSP) and the leafhopper vector Dalbulus maidis (Hemiptera: Cicadellidae), two research lines were addressed. The first one aimed to determine the indirect effects of maize infection by MBSP on some biological and behavioral parameters of the vector, whereas a second line investigated direct interactions of the phytoplasma with D. maidis during its movement through the vector body following acquisition from plants, and associated microbiota. Indirect effects were investigated in choice experiments in which alighting and oviposition preferences by D. maidis were compared on healthy vs. MBSP-infected plants with variable incubation time (diseased plants with early and advanced symptoms, or still asymptomatic). Likewise, indirect effect of MBSP on the D. maidis biology was determined in two life table experiments in which the vector was reared on healthy vs. MBSP-infected plants expressing advanced disease symptoms or still asymptomatic. Choice experiments showed that alighting and oviposition preferences of D. maidis on MBSP-infected plants compared to healthy plants depend on the pathogen incubation period in the plant. The leafhopper preferred MBSP-infected plants over healthy ones during the asymptomatic phase of the disease, but rejected infected plants with advanced symptoms. The vector was able to acquire MBSP from asymptomatic infected plants shortly (3 days) after inoculation, but transmission efficiency increased when acquisition occurred at later stages of the pathogen incubation period (≥14 days) in the source plants and the test plants showed disease symptoms faster. These results suggest that MBSP modulates D. maidis preference for asymptomatic infected plants in the early stages of the crop, allowing rapid spread of this pathogen. Maize infection by the phytoplasma had a neutral effect on most life table parameters of D. maidis; a lower net reproductivity rate (Ro) was observed in the cohort reared on MBSP-infected plants with advanced symptoms, which was compensated to some extent by a higher sexual ratio. MBSP acquisition by all vector nymphal stadia was confirmed by PCR, and the pathogen as detected in both male and female reproductive organs. Concerning direct MBSP-vector interactions, transmission electron microscopy analyses showed phytoplasma-like cells in the midgut lumen, microvilli and epithelial cells, suggesting that MBSP enters the epithelium midgut through the microvilli wall. Within the epithelial cells, mitochondria and bacteria-like cells (possibly endosymbionts) were observed together with masses of phythoplasma-like cells. In the hemocoel, phytoplasma-like cells grouped into a matrix were also observed in association with bacteria-like cells similar to those observed in the midgut epithelium. Similar associations were found in the salivary gland. Interestingly, in-situ hybridization (FISH) technique revealed a variation in diversity and abundance of the microbiota in intestine and salivary glands of D. maidis adults over time after MBSP acquisition from plants. Sulcia sp., Cardinium sp. and eubacteria increased their abundance over time, whereas Rickettsia sp. decreased. The frequent association of the vector microbiota with the phytoplasma in some tissues of D. maidis suggests that endosymbiotic bacteria may play some role in MBSP-vector interactions.

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A viral vector system was developed based on a DI-RNA, a sub-viral particle derived from TBSV-BS3-statice. This newly designed vector system was tested for its applicability in protein expression and induction of gene silencing. Two strategies were pursued. The first strategy was replication of the DI-RNA by a transgenically expressed TBSV replicase and the second was the replication by a so called helper virus. It could be demonstrated by northern blot analysis that the replicase, expressed by the transgenic N. benthamiana plant line TR4 or supplied by the helper virus, is able to replicate DI-RNA introduced into the plant cells. Various genes were inserted into different DI constructs in order to study the vector system with regard to protein expression. However, independent of how the replicase was provided no detectable amounts of protein were produced in the plants. Possible reasons for this failure are identified: the lack of systemic movement of the DI-RNA in the transgenic TR4 plants and the occurrence of deletions in the inserted genes in both systems. As a consequence the two strategies were considered unsuitable for protein expression. The DI-RNA vector system was able to induce silencing of transgenes as well as endogenous genes. Several different p19 deficient helper virus constructs were made to evaluate their silencing efficiency in combination with our DI-RNA constructs. However, it was found that our vector system can not compete with other existing VIGS (virus induced gene silencing) systems in this field. Finally, the influence of DI sequences on mRNA stability on transient GUS expression experiments in GUS silenced plants was evaluated. The GUS reporter gene system was found to be unsuitable for distinguishing between expression levels of wild type plants and GUS silenced transgenic plants. The results indicate a positive effect of the DI sequences on the level of protein expression and therefore further research into this area is recommended.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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The genomic sequences of several RNA plant viruses including cucumber mosaic virus, brome mosaic virus, alfalfa mosaic virus and tobacco mosaic virus have become available recently. The former two viruses are icosahedral while the latter two are bullet and rod shaped, respectively in particle morphology. The non-structural 3a proteins of cucumber mosaic virus and brome mosaic virus have an amino acid sequence homology of 35% and hence are evolutionarily related. In contrast, the coat proteins exhibit little homology, although the circular dichroism spectrum of these viruses are similar. The non-coding regions of the genome also exhibit variable but extensive homology. Comparison of the brome mosaic virus and alfalfa mosaic virus sequences reveals that they are probably related although with a much larger evolutionary distance. The polypeptide folds of the coat protein of three biologically distinct isometric plant viruses, tomato bushy stunt virus, southern bean mosaic virus and satellite tobacco necrosis virus have been shown to display a striking resemblance. All of them consist of a topologically similar 8-standard β-barrel. The implications of these studies to the understanding of the evolution of plant viruses will be discussed.

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Expression of antibodies in plant against essential viral proteins could provide an alternative approach to engineered viral resistance. Engineered single chain Fv antibodies scFV are particularly suitable for expression in plant because of their small size and the lack of assembly requirements. RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of viral replicases required for the replication of all positive strand RNA viruses. By using Phage technology we selected scFvs from a phage library using purified E.coli expressed TBSV(Tomato bushy stunt virus) replicase as antigen. The scFvs mediated-inhibition of RdRp activity was studied in vitro and in planta. In vitro experiments showed the inhibition of CNV(Cucumber necrosis virus) and TCV(Turnip crinkle virus) RdRp. Transient in planta assays based on agroinfiltration and an infectious clone of TBSV demonstrated the inhibition of the replication of TBSV(Tomato bushy stunt virus). Epitope mapping showed that the selected scFvs target the motif E of RdRp which is involved in template binding.Moreover T1 plants of transgenic lines of N. benthamiana expressing different scFvs either in the cytoplasm or the ER (endoplasmic reticulum) showed a high level of resistance against infection with TBSV and RCNMV(Red clover necrotic mosaic virus) upon inoculation with virus particles. This is the first report that scFvs against a RdRp of a plant viruses can inhibit viral replication in vivo. The resistance is even efficient against viruses belonging to different virus families.

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Each list is an addendum to the previous list.

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Nitrous oxide (N2O) is a major greenhouse gas (GHG) product of intensive agriculture. Fertilizer nitrogen (N) rate is the best single predictor of N2O emissions in row-crop agriculture in the US Midwest. We use this relationship to propose a transparent, scientifically robust protocol that can be utilized by developers of agricultural offset projects for generating fungible GHG emission reduction credits for the emerging US carbon cap and trade market. By coupling predicted N2O flux with the recently developed maximum return to N (MRTN) approach for determining economically profitable N input rates for optimized crop yield, we provide the basis for incentivizing N2O reductions without affecting yields. The protocol, if widely adopted, could reduce N2O from fertilized row-crop agriculture by more than 50%. Although other management and environmental factors can influence N2O emissions, fertilizer N rate can be viewed as a single unambiguous proxy—a transparent, tangible, and readily manageable commodity. Our protocol addresses baseline establishment, additionality, permanence, variability, and leakage, and provides for producers and other stakeholders the economic and environmental incentives necessary for adoption of agricultural N2O reduction offset projects.

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Rice grassy stunt virus is a member of the genus Tenuivirus, is persistently transmitted by a brown planthopper, and has occurred in rice plants in South, Southeast, and East Asia (similar to North and South America). We determined the complete nucleotide (nt) sequences of RNAs 1 (9760 nt), 2 (4069 nt), 3 (3127 nt), 4 (2909 nt), 5 (2704 nt), and 6 (2590 nt) of a southern Philippine isolate from South Cotabato and compared them with those of a northern Philippine isolate from Laguna (Toriyama et al., 1997, 1998). The numbers of nucleotides in the terminal untranslated regions and open reading frames were identical between the two isolates except for the 5′ untranslated region of the complementary strand of RNA 4. Overall nucleotide differences between the two isolates were only 0.08% in RNA 1, 0.58% in RNA 4, and 0.26% in RNA 5, whereas they were 2.19% in RNA 2, 8.38% in RNA 3, and 3.63% in RNA 6. In the intergenic regions, the two isolates differed by 9.12% in RNA 2, 11.6% in RNA 3, and 6.86% in RNA 6 with multiple consecutive nucleotide deletion/insertions, whereas they differed by only 0.78% in RNA 4 and 0.34% in RNA 5. The nucleotide variation in the intergenic region of RNA 6 within the South Cotabato isolate was only 0.33%. These differences in accumulation of mutations among individual RNA segments indicate that there was genetic reassortment in the two geographical isolates; RNAs 1, 4, and 5 of the two isolates came from a common ancestor, whereas RNAs 2, 3, and 6 were from two different ancestors.

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Agricultural soils emit about 50% of the global flux of N2O attributable to human influence, mostly in response to nitrogen fertilizer use. Recent evidence that the relationship between N2O fluxes and N-fertilizer additions to cereal maize are non-linear provides an opportunity to estimate regional N2O fluxes based on estimates of N application rates rather than as a simple percentage of N inputs as used by the Intergovernmental Panel on Climate Change (IPCC). We combined a simple empirical model of N2O production with the SOCRATES soil carbon dynamics model to estimate N2O and other sources of Global Warming Potential (GWP) from cereal maize across 19,000 cropland polygons in the North Central Region (NCR) of the US over the period 1964–2005. Results indicate that the loading of greenhouse gases to the atmosphere from cereal maize production in the NCR was 1.7 Gt CO2e, with an average 268 t CO2e produced per tonne of grain. From 1970 until 2005, GHG emissions per unit product declined on average by 2.8 t CO2e ha−1 annum−1, coinciding with a stabilisation in N application rate and consistent increases in grain yield from the mid-1970’s. Nitrous oxide production from N fertilizer inputs represented 59% of these emissions, soil C decline (0–30 cm) represented 11% of total emissions, with the remaining 30% (517 Mt) from the combustion of fuel associated with farm operations. Of the 126 Mt of N fertilizer applied to cereal maize from 1964 to 2005, we estimate that 2.2 Mt N was emitted as N2O when using a non-linear response model, equivalent to 1.75% of the applied N.

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Crop simulation models have the potential to assess the risk associated with the selection of a specific N fertilizer rate, by integrating the effects of soil-crop interactions on crop growth under different pedo-climatic and management conditions. The objective of this study was to simulate the environmental and economic impact (nitrate leaching and N2O emissions) of a spatially variable N fertilizer application in an irrigated maize field in Italy. The validated SALUS model was run with 5 nitrogen rates scenarios, 50, 100, 150, 200, and 250 kg N ha−1, with the latter being the N fertilization adopted by the farmer. The long-term (25 years) simulations were performed on two previously identified spatially and temporally stable zones, a high yielding and low yielding zone. The simulation results showed that N fertilizer rate can be reduced without affecting yield and net return. The marginal net return was on average higher for the high yield zone, with values ranging from 1550 to 2650 € ha−1 for the 200 N and 1485 to 2875 € ha−1 for the 250 N. N leaching varied between 16.4 and 19.3 kg N ha−1 for the 200 N and the 250 N in the high yield zone. In the low yield zone, the 250 N had a significantly higher N leaching. N2O emissions varied between 0.28 kg N2O ha−1 for the 50 kg N ha−1 rate to a maximum of 1.41 kg N2O ha−1 for the 250 kg N ha−1 rate.

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Maize streak virus (MSV; family Geminiviridae, genus Mastrevirus), the causal agent of maize streak disease, ranks amongst the most serious biological threats to food security in subSaharan Africa. Although five distinct MSV strains have been currently described, only one of these - MSV-A - causes severe disease in maize. Due primarily to their not being an obvious threat to agriculture, very little is known about the 'grass-adapted' MSV strains, MSV-B, -C, -D and -E. Since comparing the genetic diversities, geographical distributions and natural host ranges of MSV-A with the other MSV strains could provide valuable information on the epidemiology, evolution and emergence of MSV-A, we carried out a phylogeographical analysis of MSVs found in uncultivated indigenous African grasses. Amongst the 83 new MSV genomes presented here, we report the discovery of six new MSV strains (MSV-F to -K). The non-random recombination breakpoint distributions detectable with these and other available mastrevirus sequences partially mirror those seen in begomoviruses, implying that the forces shaping these breakpoint patterns have been largely conserved since the earliest geminivirus ancestors. We present evidence that the ancestor of all MSV-A variants was the recombinant progeny of ancestral MSV-B and MSV-G/-F variants. While it remains unknown whether recombination influenced the emergence of MSV-A in maize, our discovery that MSV-A variants may both move between and become established in different regions of Africa with greater ease, and infect more grass species than other MSV strains, goes some way towards explaining why MSV-A is such a successful maize pathogen. © 2008 SGM.

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Maize streak virus (MSV) contributes significantly to the problem of extremely low African maize yields. Whilst a diverse range of MSV and MSV-like viruses are endemic in sub-Saharan Africa and neighbouring islands, only a single group of maize-adapted variants - MSV subtypes A1 -A6 - causes severe enough disease in maize to influence yields substantially. In order to assist in designing effective strategies to control MSV in maize, a large survey covering 155 locations was conducted to assess the diversity, distribution and genetic characteristics of the Ugandan MSV-A population. PCR-restriction fragment-length polymorphism analyses of 391 virus isolates identified 49 genetic variants. Sixty-two full-genome sequences were determined, 52 of which were detectably recombinant. All but two recombinants contained predominantly MSV-A1-like sequences. Of the ten distinct recombination events observed, seven involved inter-MSV-A subtype recombination and three involved intra-MSV-A1 recombination. One of the intra-MSV-A1 recombinants, designated MSV-A1 UgIII, accounted for >60% of all MSV infections sampled throughout Uganda. Although recombination may be an important factor in the emergence of novel geminivirus variants, it is demonstrated that its characteristics in MSV are quite different from those observed in related African cassava-infecting geminivirus species. © 2007 SGM.

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Geminivirus infectivity is thought to depend on interactions between the virus replication-associated proteins Rep or RepA and host retinoblastoma-related proteins (pRBR), which control cell-cycle progression. It was determined that the substitution of two amino acids in the Maize streak virus (MSV) RepA pRBR-interaction motif (LLCNE to LLCLK) abolished detectable RepA-pRBR interaction in yeast without abolishing infectivity in maize. Although the mutant virus was infectious in maize, it induced less severe symptoms than the wild-type virus. Sequence analysis of progeny viral DNA isolated from infected maize enabled detection of a high-frequency single-nucleotide reversion of C(601)A in the 3 nt mutated sequence of the Rep gene. Although it did not restore RepA-pRBR interaction in yeast, sequence-specific PCR showed that, in five out of eight plants, the C(601)A reversion appeared by day 10 post-inoculation. In all plants, the C(601)A revertant eventually completely replaced the original mutant population, indicating a high selection pressure for the single-nucleotide reversion. Apart from potentially revealing an alternative or possibly additional function for the stretch of DNA that encodes the apparently non-essential pRBR-interaction motif of MSV Rep, the consistent emergence and eventual dominance of the C(601)A revertant population might provide a useful tool for investigating aspects of MSV biology, such as replication, mutation and evolution rates, and complex population phenomena, such as competition between quasispecies and population turnover. © 2005 SGM.