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Reverse transcription-quantitative PCR (RT-qPCR) is an analytical tool for gene expression quantification. Reference genes are not yet available for gene expression analysis during interactions of Ralstonia solanacearum with 'Hawaii 7996' (the most stable source of resistance in tomato). Here, we carried out a multi-algorithm stability analysis of eight candidate reference genes during interactions of 'Hawaii 7996' with one incompatible/avirulent and two compatible/virulent (= resistance-breaking) bacterial isolates. Samples were taken at 24- and 96-h post-inoculation (HPI). Analyses were performed using the ∆∆Ct method and expression stability was estimated using BestKeeper, NormFinder, and geNorm algorithms. TIP41 and EF1α (with geNorm), TIP41 and ACT (with NormFinder), and UBI3 and TIP41 (with BestKeeper), were the best combinations for mRNA normalization in incompatible interactions at 24 HPI and 96 HPI. The most stable genes in global compatible and incompatible interactions at 24 HPI and 96 HPI were PDS and TIP41 (with geNorm), TIP41 and ACT (with NormFinder), and UBI3 and PDS/EXP (with BestKeeper). Global analyses on the basis of the three algorithms across 20 R. solanacearum-tomato experimental conditions identified UBI3, TIP41 and ACT as the best choices as reference tomato genes in this important pathosystem.

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BACKGROUND: Silver nanoparticles (AgNPs), particularly those entrapped in polymeric nanosystems, have arisen as options for managing plant bacterial diseases. Among the biopolymers useful for the entrapment of AgNPs, chitosan is promising because of its low cost, good biocompatibility, antimicrobial properties and biodegradability. The present study aimed: (i) to greenly-synthesize AgNPs using different concentrations of aqueous extract of tomato leaves followed by entrapment of AgNPs with chitosan (CH-AgNPs); (ii) to characterize the optical, structural and biological properties of the nanosystems produced; (iii) to evaluate the antimicrobial activities of AgNPs and nanomaterials; and (iv) to assess the effectiveness of AgNPs and nanomaterials for controlling tomato bacterial wilt caused by Ralstonia solanacearum. RESULTS: Spherical and oval AgNPs had incipient colloidal instability, although the concentration of the tomato leaf extract influenced both size (< 87 nm) and the polydispersity index. Nanomaterials (< 271 nm in size) were characterized by a highly stable matrix of chitosan containing polydisperse AgNPs. Free AgNPs and CH-AgNPs were stable for up to 30 days, with no significant alteration in physicochemical parameters. The AgNPs and nanomaterials had antibacterial activity and decreased bacterial growth at micromolar concentrations after 48 h. Morphological changes in R. solanacearum cells were observed after treatment with CH-AgNPs. The application of CH-AgNPs at 256 µmol L-1 reduced the incidence of bacterial wilt in a partially resistant tomato genotype but not in the susceptible line. CONCLUSION: Greenly-synthesized chitosan-derived nanomaterials containing AgNPs produced with leaf extracts from their own species appear to comprise a promising and sustainable alternative in an integrated management approach aiming to reduce the yield losses caused by bacterial wilt. © 2019 Society of Chemical Industry.

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Bacterial wilt induced by the Ralstonia solanacearum species complex is endemic to Brazil, where it can cause variable losses in many hosts. Its economic importance, however, cannot be precisely measured due to Brazil's continental size, subject to variable weather conditions which directly affect disease expression. The objectives of this paper were (i) to gather scattered information on historical facts; (ii) to show the current distribution of the pathogen in the country, and (iii) to comment on future trends on the importance of the disease in economically important current and potential hosts, based on the pathogen's variability and the global climate change under way.

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We report here the complete genome sequences of two Ralstonia pseudosolanacearum strains, isolated from the warm northeast region of Brazil. They display divergent (compatible versus incompatible) interactions with the resistant tomato line Hawaii 7996. Polymorphisms were detected in a subset of effector genes that might be associated with these contrasting phenotypes.

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The objective of the current study was to isolate and identify phenotypes of Staphylococcus spp. strains derived from buffalo (Bubalus bubalis) milk. A total of 548 milk samples from 137 buffalo were cultured in Columbia agar enriched with 5% defibrinated sheep blood. Determination of the capacity of Staphylococcus aureus to produce enterotoxins A-D and toxic shock syndrome toxin-1 (TSST-1) was achieved by reverse passive latex agglutination (RPLA). Antimicrobial sensitivity of S. aureus strains was evaluated using the disk diffusion technique, and ß-lactamase detection was achieved using the chromogenic test with paper discs impregnated with nitrocefin. From all the mammary quarters examined, 36 (10.8%) were positive for Staphylococcus spp., 83.3% were coagulase-negative staphylococcus (CNS), 11.1% were coagulase-positive staphylococcus (CPS), and 5.6% were of CPS+CNS positive. All isolates of S. aureus produced at least 1 toxin and 5 out of 6 isolates (83.0%) produced ß-lactamase. One hundred percent of S. aureus isolates were sensitive to methicillin and amoxicillin + clavulanic acid, and resistant to ampicillin, penicillin, and oxacillin. Analysis of the results obtained in the current study highlight the epidemiologic importance of buffalo milk regarding the production of enterotoxins and TSST-1 and the potential risk to public health.

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Objetivo: avaliar a significância clínica de estafilococos coagulase-negativa (ECN) isolados de processos infecciosos em recém-nascidos da unidade neonatal do Hospital das Clínicas da Faculdade de Medicina de Botucatu.Método: as linhagens de ECN isoladas foram identificadas eclassificadas em significativas e contaminantes, com base em uma série de dados clínicos e laboratoriais obtidos dos prontuários dos pacientes internados na unidade neonatal. Foram pesquisados os dados referentes a fatores perinatais de risco para infecção, evolução clínica, alterações do hemograma e/ou positividade de proteína C-reativa e antibioticoterapia.Resultados: das 117 linhagens de ECN isoladas, 60 (51,3por cento)foram classificadas como significativas, e 57 (48,7por cento) como contaminantes. Das 54 crianças com infecção por ECN, 43 (79,6por cento) eram prematuras, e 27 (50,0por cento) tiveram peso ao nascimento < 1.500g. A maioria das crianças com infecção por ECN estava submetida a dois ou mais procedimentos invasivos (77,8por cento), incluindo o uso de cateter(88,9por cento), nutrição parenteral (64,8por cento) e ventilação mecânica (61,1por cento).O S.epidermidis foi a espécie mais freqUentemente isolada (77,8por cento), e mais associada com infecção (86,7por cento) do que com contaminação (68,4por centi). Outras espécies de ECN, incluindo duas linhagens de s.haemolyticus, três linhagens de s. lugdunensis, uma linhagem de S. simulans, uma de S. warneri e uma linhagem de S. xylosus também foram isoladas de crianças com evidência clínica de pneumonia, nterocolite necrosante e sepse.Conclusão: a maioria dos recém-nascidos com infecção por ECN apresentou...

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OBJECTIVE: To evaluate the clinical significance of coagulase-negative staphylococci (CNS) isolated from newborns' infections at Neonatal Unit of Hospital das Clínicas da Faculdade de Medicina de Botucatu. METHODS: The CNS strains isolated were identified and classified as clinically significant and contaminant, based on a series of clinical and laboratory data obtained from patients who stayed in the Neonatal Unit. The following data were analyzed: risk factors for infections, clinical evolution, abnormal blood cell counts and/or C-reactive protein and antibiotic therapy. RESULTS: Among the 117 CNS strains isolated, 60 (51.3%) were classified as significant and 57 (48.7%) as contaminant. Among the 54 infants infected by CNS, 43 (79.6%) presented very low birthweight (<1,500 g). Most of the infants infected by CNS were submitted to two or more invasive procedures (77.8%), including use of catheter (88.9%), parenteral nutrition (64.8%) and mechanical ventilation (61.1%). Staphylococcus epidermidis was the most frequently isolated species (77.8%) and more often associated with infection (86.7%) than with contamination (68.4%). Other species of CNS, including two strains of S. haemolyticus, three strains of S. lugdunensis, one strain of S. simulans, one strain of S. warneri and one strain of S. xylosus were also isolated from infants with clinical evidence of pneumonia, necrotizing enterocolitis and sepsis. CONCLUSIONS: Most newborns infected by CNS presented important risk factors for infection onset, including birthweight <1,500 g, foreign body presence and previous use of antibiotics. The identification of CNS species constitutes a useful marker of infection, since S. epidermidis was the species more frequently associated with infection.