It is well known that citrus plants that have been infected by Xylella fastidiosa display nutritional deficiencies, probably caused by production of extracellular polymers by the bacteria that block normal nutrient flow through the xylem. The aim of this work was to study the mineral composition of specific foliar areas in different stages of infection in citrus. Thus, the concentrations of macro and micronutrients in leaves of citrus infected by X. fastidiosa were measured. Samples from four infected citrus orchards in the State of São Paulo, Brazil, were respectively collected from Santa Rita do Passa Quatro, Neves Paulista, Gavião Peixoto and Paraíso counties. The presence of X. fastidiosa in leaves was confirmed by polymerase chain reaction (PCR) using specific PCR primers. To understand the variation in leaf-nutrient content in citrus plants, we used foliar nutrient values from control (non-symptomatic) plants as a reference. Chemometric analysis showed that the deficiency of P and K in symptomatic trees for all orchards and high concentrations of Fe, Mn and Zn were observed in chlorotic areas, although other studies revealed deficiency of zinc in leaves. This is the first report showing that a correlation between chlorotic citrus leaf and higher concentrations of Fe, Mn and Zn are observed when infected and healthy plants were compared.

Microbial community composition was examined in two soil types, Anthrosols and adjacent soils, sampled from three locations in the Brazilian Amazon. The Anthrosols, also known as Amazonian dark earths, are highly fertile soils that are a legacy of pre-Columbian settlement. Both Anthrosols and adjacent soils are derived from the same parent material and subject to the same environmental conditions, including rainfall and temperature; however, the Anthrosols contain high levels of charcoal-like black carbon from which they derive their dark color. The Anthrosols typically have higher cation exchange capacity, higher pH, and higher phosphorus and calcium contents. We used culture media prepared from soil extracts to isolate bacteria unique to the two soil types and then sequenced their 16S rRNA genes to determine their phylogenetic placement. Higher numbers of culturable bacteria, by over two orders of magnitude at the deepest sampling depths, were counted in the Anthrosols. Sequences of bacteria isolated on soil extract media yielded five possible new bacterial families. Also, a higher number of families in the bacteria were represented by isolates from the deeper soil depths in the Anthrosols. Higher bacterial populations and a greater diversity of isolates were found in all of the Anthrosols, to a depth of up to 1 m, compared to adjacent soils located within 50-500 m of their associated Anthrosols. Compared to standard culture media, soil extract media revealed diverse soil microbial populations adapted to the unique biochemistry and physiological ecology of these Anthrosols.

This study describes a rapid procedure for the isolation of genomic DNA from Staphylococcus aureus that yielded a good amount of high quality DNA for the amplification of staphylococcal enterotoxins genes (A, B, C, D, and E) and the TSST-1 gene as well as enzymatic restriction (HaeIII) from environmental isolates. With this method, it was possible to detect these genes in a sample containing as little as 10(5) cells with positive PCR reactions obtained from approximately 10pg of DNA in a final reaction volume of 25µl.Descreve-se um procedimento rápido para extração de DNA genômico de isolados de Staphylococcus aureus capaz de produzir DNA estafilocócico em qualidade e quantidade suficiente para a amplificação de genes que codificam enterotoxinas estafilocócicas (A - E) e para TSST-1 e restrição enzimática (HaeIII) de isolados ambientais. O método proposto foi capaz de detectar esses genes em um produto de extração contendo tanto quanto 10(5) células, e reações positivas de PCR foram obtidas de aproximadamente 10pg de DNA.

This study describes a rapid procedure for the isolation of genomic DNA from Staphylococcus aureus that yielded a good amount of high quality DNA for the amplification of staphylococcal enterotoxins genes (A, B, C, D, and E) and the TSST-1 gene as well as enzymatic restriction (HaeIII) from environmental isolates. With this method, it was possible to detect these genes in a sample containing as little as 10(5) cells with positive PCR reactions obtained from approximately 10pg of DNA in a final reaction volume of 25µl.

Bacterial production of siderophores may involve specific genes related to nonribosomal peptide and polyketide biosynthesis, which have not been fully identified in the genome of Xylella fastidiosa strain 9a5c. However, a search for siderophore-related genes in strain 9a5c indicated five membrane receptors, including siderophore, ferrichrome-iron and hemin receptors. All these biomolecules are thought to be associated with iron transport and utilization. Eighty isolates obtained from citrus orchards containing trees that developed citrus variegated chlorosis (CVC) were screened for siderophore production. The results demonstrated that only 10 of the isolates did not produce siderophores. Additional strains obtained from coffee, almond, mulberry, elm, ragweed, periwinkle and grape also infected by X. fastidiosa were also shown by the chromeazurol bioassay to produce siderophores. In order to correlate siderophore production with the presence of siderophore-related genes, a polymerase chain reaction (PCR) was developed using specific primers for the catechol-type ferric enterobactin receptor (pfeA) and the hydroxamate-type ferrisiderophore receptor (fiuA) genes of strain 9a5c. The PCR results confirmed our hypothesis by demonstrating that amplification products were detected in all strains except for those isolates that did not produce siderophores.

ABSTRACT During a 2-year period (2003-2004), 132 strains of Staphylococcusaureus isolated from crude milk (without thermal treatment) collected in different places in Piracicaba, São Paulo State, Brazil, were investigated for the presence of genes for enterotoxins (ent) and toxic shock syndrome toxin-1 (tst). Polymerase-chain reaction (PCR) was performed by using 6 pairs of relevant oligonucleotide primers. Ninety isolates (68.18%) were positive for (47 strains) or 2 (43 strains) toxin genes. The combination of entA and tst showed the highest prevalence (33 strains).The good correlation between PCR results and toxin protein detection and identification by optimum-sensitivity-plate (OSP) test was observed when 44.45% of strains showed positive for toxin production.

ABSTRACT The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium Xylella fastidiosa. The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates. Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the gyrB gene were determined for 18 strains of X. fastidiosa isolated from different plant hosts. A phylogenetic analysis, based on gyrB , grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains. Primer pairs designed for the 16S rRNA and gyrB genes were extensively searched in databases to verify their in silico specificity. Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity. A multiplex PCR protocol was developed and its sensitivity tested. Sequencing of PCR products confirmed the validity of the multiplex PCR. Xylella fastidiosa was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants. Specific detection of X. fastidiosa may facilitate the understanding of its ecological significance and prevention of spread of the disease.

The metabolic activities of root nodules formed by Rhizobium tropici UM1899 were measured to test for the effects of geographical origin of the host bean (Phaseolus vulgaris L.) plant. Under increasing levels of N (0 to 24 mM of NH4NO3), the optimum condition for nitrogen fixation based on nitrogenase activity and allantoin concentration, was obtained between 2 and 4 mM N. Cultivars, including wild accessions from the two major domestication centers in America (Middle America and Andes), were then grown under aseptic conditions with 2 mM NH4NO3 and the rhizobial inoculant. Plant nodulins [leghaemoglobin (Lb), phosphoenolypyruvate carboxylase (PEPC) and glutamine synthetase (GS)], bacterial nitrogenase (NIF) activities as well as allantoin (ALA) concentration in the xylem sap, were assayed in flowering plants. Lb, PEPC, NIF activities and ALA concentrations were strongly affected by cultivar and by the center of origin. GS activity did not vary significantly with either cultivar or center of origin. LB, NIF and ALA were directly related to plant growth and offer opportunities to select for efficient N2-fixing symbioses. There were slight increases in nodulin activities of the domesticated cultivars, but the overall low variability within this material relative to landraces suggests that diversity for biological nitrogen fixation was reduced by domestication.