Caracterização de isolados e reação de Capsicum spp. ao Cucumber mosaic virus (CMV)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Breakdown of resistance in sweet pepper against Pepper yellow mosaic virus in Brazil

Plantas de Capsicum annuum cv. Magali R, resistentes ao Pepper yellow mosaic virus (PepYMV), exibindo sintomas severos de mosaico amarelo, malformação foliar e subdesenvolvimento foram encontradas em plantios na região de Lins, SP, Brasil, em 2003/04. Partículas semelhantes àquelas do gênero Potyvirus foram observadas em extrato foliar de planta infectada examinado em microscópio eletrônico de transmissão. O extrato foliar também reagiu com anti-soro contra o PepYMV em PTA-ELISA. Além de C. annuum cv. Magali R, esse potyvirus também infectou sistemicamente C. annuum cv. Rubia R, que é resistente ao PepYMV. A seqüência de nucleotídeos de parte do gene da proteína capsidial (CP) desse potyvirus apresentou 96-98% de identidade com a de outros isolados do PepYMV. A seqüência parcial de nucleotídeos da região 3' não traduzida (3' NTR) apresentou 94-96% de identidade com a do PepYMV. Esses resultados são indicativos de que o potyvirus que quebrou a resistência em pimentão é um isolado do PepYMV.

Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato

The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site–leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species.

Targeting nuclear factor-kappa B to overcome resistance to chemotherapy

Intrinsic or acquired resistance to chemotherapeutic agents is a common phenomenon and a major challenge in the treatment of cancer patients. Chemoresistance is defined by a complex network of factors including multi-drug resistance proteins, reduced cellular uptake of the drug, enhanced DNA repair, intracellular drug inactivation, and evasion of apoptosis. Pre-clinical models have demonstrated that many chemotherapy drugs, such as platinum-based agents, antracyclines, and taxanes, promote the activation of the NF-κB pathway. NF-κB is a key transcription factor, playing a role in the development and progression of cancer and chemoresistance through the activation of a multitude of mediators including anti-apoptotic genes. Consequently, NF-κB has emerged as a promising anti-cancer target. Here, we describe the role of NF-κB in cancer and in the development of resistance, particularly cisplatin. Additionally, the potential benefits and disadvantages of targeting NF-κB signaling by pharmacological intervention will be addressed.

Using simulation techniques to investigate methods to determine resistance of helminths to anthelmintic treatment

The widespread and increasing resistance of internal parasites to anthelmintic control is a serious problem for the Australian sheep and wool industry. As part of control programmes, laboratories use the Faecal Egg Count Reduction Test (FECRT) to determine resistance to anthelmintics. It is important to have confidence in the measure of resistance, not only for the producer planning a drenching programme but also for companies investigating the efficacy of their products. The determination of resistance and corresponding confidence limits as given in anthelmintic efficacy guidelines of the Standing Committee on Agriculture (SCA) is based on a number of assumptions. This study evaluated the appropriateness of these assumptions for typical data and compared the effectiveness of the standard FECRT procedure with the effectiveness of alternative procedures. Several sets of historical experimental data from sheep and goats were analysed to determine that a negative binomial distribution was a more appropriate distribution to describe pre-treatment helminth egg counts in faeces than a normal distribution. Simulated egg counts for control animals were generated stochastically from negative binomial distributions and those for treated animals from negative binomial and binomial distributions. Three methods for determining resistance when percent reduction is based on arithmetic means were applied. The first was that advocated in the SCA guidelines, the second similar to the first but basing the variance estimates on negative binomial distributions, and the third using Wadley’s method with the distribution of the response variate assumed negative binomial and a logit link transformation. These were also compared with a fourth method recommended by the International Co-operation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) programme, in which percent reduction is based on the geometric means. A wide selection of parameters was investigated and for each set 1000 simulations run. Percent reduction and confidence limits were then calculated for the methods, together with the number of times in each set of 1000 simulations the theoretical percent reduction fell within the estimated confidence limits and the number of times resistance would have been said to occur. These simulations provide the basis for setting conditions under which the methods could be recommended. The authors show that given the distribution of helminth egg counts found in Queensland flocks, the method based on arithmetic not geometric means should be used and suggest that resistance be redefined as occurring when the upper level of percent reduction is less than 95%. At least ten animals per group are required in most circumstances, though even 20 may be insufficient where effectiveness of the product is close to the cut off point for defining resistance.

Methods to determine resistance to anthelmintics when continuing larval development occurs

The in vivo faecal egg count reduction test (FECRT) is the most commonly used test to detect anthelmintic resistance (AR) in gastrointestinal nematodes (GIN) of ruminants in pasture based systems. However, there are several variations on the method, some more appropriate than others in specific circumstances. While in some cases labour and time can be saved by just collecting post-drench faecal worm egg counts (FEC) of treatment groups with controls, or pre- and post-drench FEC of a treatment group with no controls, there are circumstances when pre- and post-drench FEC of an untreated control group as well as from the treatment groups are necessary. Computer simulation techniques were used to determine the most appropriate of several methods for calculating AR when there is continuing larval development during the testing period, as often occurs when anthelmintic treatments against genera of GIN with high biotic potential or high re-infection rates, such as Haemonchus contortus of sheep and Cooperia punctata of cattle, are less than 100% efficacious. Three field FECRT experimental designs were investigated: (I) post-drench FEC of treatment and controls groups, (II) pre- and post-drench FEC of a treatment group only and (III) pre- and post-drench FEC of treatment and control groups. To investigate the performance of methods of indicating AR for each of these designs, simulated animal FEC were generated from negative binominal distributions with subsequent sampling from the binomial distributions to account for drench effect, with varying parameters for worm burden, larval development and drench resistance. Calculations of percent reductions and confidence limits were based on those of the Standing Committee for Agriculture (SCA) guidelines. For the two field methods with pre-drench FEC, confidence limits were also determined from cumulative inverse Beta distributions of FEC, for eggs per gram (epg) and the number of eggs counted at detection levels of 50 and 25. Two rules for determining AR: (1) %reduction (%R) < 95% and lower confidence limit <90%; and (2) upper confidence limit <95%, were also assessed. For each combination of worm burden, larval development and drench resistance parameters, 1000 simulations were run to determine the number of times the theoretical percent reduction fell within the estimated confidence limits and the number of times resistance would have been declared. When continuing larval development occurs during the testing period of the FECRT, the simulations showed AR should be calculated from pre- and post-drench worm egg counts of an untreated control group as well as from the treatment group. If the widely used resistance rule 1 is used to assess resistance, rule 2 should also be applied, especially when %R is in the range 90 to 95% and resistance is suspected.

Evaluation of resistance to Tomato severe rugose virus (ToSRV) in Capsicum spp. genotypes

Tomato severe rugose virus (ToSRV) is the predominant species of begomovirus in São Paulo State, Brazil, and infects primarily tomato and pepper plants. There is no information about genetic resistance of pepper to this virus, so in this work the reaction of 29 genotypes of Capsicum spp. was evaluated by inoculation of two ToSRV isolates: ToSRV-Sk (isolated from a tomato plant) and ToSRV-PJU (isolated from a pepper plant). For both isolates, two C. annuun genotypes (Catarino Cascabel - México and Silver) showed no symptoms 30 days after inoculation (d.a.i). In a second experiment, these two genotypes were evaluated for 150 d.a.i and, again, no symptoms could be observed. However, the virus was detected by RCA-PCR, indicating that both genotypes are susceptible, but less affected by ToSRV infection. Catarino Cascabel - México and Silver can be indicated for use in breeding programs for resistance of pepper to ToSRV.

A classification of Pepper yellow mosaic virus isolates into pathotypes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Peroxidase enzyme (EC.1.11.1.7) activity as an indicator of water stress in sweet pepper plants

The present work was carried out at the Faculdade de Ciências Agronômicas - UNESP, Botucatu, SP. The purpose of the study was to evaluate the physiological and biochemical behavior of sweet pepper (Capsicum annuum L.) plants under different soil water availability conditions and the efficiency of the peroxidase (EC. 1.11.1.7) activity as an indicator of water stress in plants. Sweet pepper plants were grown for 230 days after transplanting of seedlings. The experiment was arranged in a completely randomized experimental design with 4 treatments, two irrigation managements (50 and 1500 kPa) and two soil surface managements (presence or absence of black polyethylene covering), and six replications. Physiological activities, such as stomatal transpiration and resistance to water vapor diffusion, were evaluated, as well as biochemical activities, such as peroxidase activity and total soluble protein in foliar tissues. It was observed that soil water availability may lead to physiological and biochemical alterations in plants. Successive water stress cycles may promote the development of characteristics responsible for improving the plant tolerance to periods of low water availability. The peroxidase enzyme activity showed to be an efficient indicator of water stress in sweet pepper plants.

Peroxidase activity as an indicator of water stress in sweet pepper plants

The purpose of the study was to evaluate the physiological and biochemical behavior of sweet pepper (Capsicum annuum L.) plants under different soil water availability conditions and the efficiency of the peroxidase (EC. 1.11. 1.7) activity as an indicator of water stress in plants. The experiment was carried out at the Faculdade de Ciências Agronômicas UNESP, Botucatu, SP. Sweet pepper plants were grown for 230 days after transplanting of seedlings and arranged in a completely randomized experimental design with 4 treatments, two irrigation managements (50 and 1500 kPa) and two soil surface managements (presence or absence of black polyethylene covering), and six replications. Physiological activities, such as stomatal transpiration and resistance to water vapor diffusion, were evaluated as well as biochemical activities, such as peroxidase activity and total soluble protein in foliar tissues. It was observed that soil water availability may lead to physiological and biochemical alterations in plants. Successive water stress cycles may promote the development of characteristics responsible for improving plant tolerance to periods of low water availability. The peroxidase enzyme activity showed to be an efficient indicator of water stress in sweet pepper plants.

Molecular signals required for type III secretion and translocation of the Xanthomonas campestris AvrBs2 protein to pepper plants

Strains of Xanthomonas campestris pv. vesicatoria (Xcv) carrying avrBs2 are specifically recognized by Bs2 pepper plants, resulting in localized cell death and plant resistance. Agrobacterium-mediated transient expression of the Xcv avrBs2 gene in plant cells results in Bs2-dependent cell death, indicating that the AvrBs2 protein alone is sufficient for the activation of disease resistance-mediated cell death in planta. We now provide evidence that AvrBs2 is secreted from Xcv and that secretion is type III (hrp) dependent. N- and C-terminal deletion analysis of AvrBs2 has identified the effector domain of AvrBs2 recognized by Bs2 pepper plants. By using a truncated Pseudomonas syringae AvrRpt2 effector reporter devoid of type III signal sequences, we have localized the minimal region of AvrBs2 required for type III secretion in Xcv. Furthermore, we have identified the region of AvrBs2 required for both type III secretion and translocation to host plants. The mapping of AvrBs2 sequences sufficient for type III delivery also revealed the presence of a potential mRNA secretion signal.

Smoothing a Discrete Hazard Function for the Number of Patients Colonized with Methicillin-Resistance Staphylococcus Aureus in an Intensive Care Unit

A new method for estimating the time to colonization of Methicillin-resistant Staphylococcus Aureus (MRSA) patients is developed in this paper. The time to colonization of MRSA is modelled using a Bayesian smoothing approach for the hazard function. There are two prior models discussed in this paper: the first difference prior and the second difference prior. The second difference prior model gives smoother estimates of the hazard functions and, when applied to data from an intensive care unit (ICU), clearly shows increasing hazard up to day 13, then a decreasing hazard. The results clearly demonstrate that the hazard is not constant and provide a useful quantification of the effect of length of stay on the risk of MRSA colonization which provides useful insight.