Bacteria Murmur: Application of an Acoustic Biosensor for Plant Pathogen Detection.

A multi-targeting protocol for the detection of three of the most important bacterial phytopathogens, based on their scientific and economic importance, was developed using an acoustic biosensor (the Quartz Crystal Microbalance) for DNA detection. Acoustic detection was based on a novel approach where DNA amplicons were monitored and discriminated based on their length rather than mass. Experiments were performed during real time monitoring of analyte binding and in a direct manner, i.e. without the use of labels for enhancing signal transduction. The proposed protocol improves time processing by circumventing gel electrophoresis and can be incorporated as a routine detection method in a diagnostic lab or an automated lab-on-a-chip system for plant pathogen diagnostics

Environmental Spread of Antibiotic Resistance

Antibiotic resistance represents a global health concern. Soil, water, livestock and plant foods are directly or indirectly exposed to antibiotics due to their agricultural use or contamination. This selective pressure has acted synergistically to bacterial competition in nature to breed antibiotic-resistant (AR) bacteria. Research over the past few decades has focused on the emergence of AR pathogens in food products that can cause disease outbreaks and the spread of antibiotic resistance genes (ARGs), but One Health approaches have lately expanded the focus to include commensal bacteria as ARG donors. Despite the attempts of national and international authorities of developed and developing countries to reduce the over-prescription of antibiotics to humans and the use of antibiotics as livestock growth promoters, the selective flow of antibiotic resistance transmission from the environment to the clinic (and vice-versa) is increasing. This review focuses on the mechanisms of ARG transmission and the hotspots of antibiotic contamination resulting in the subsequent emergence of ARGs. It follows the transmission of ARGs from farm to plant and animal food products and provides examples of the impact of ARG flow to clinical settings. Understudied and emerging antibiotic resistance selection determinants, such as heavy metal and biocide contamination, are also discussed here

Multiplex polymerase chain reaction showing specificity of three primer pairs to amplify <i>Pseudomonas syringae</i> pv. <i>tomato</i> (lane 1–6), <i>Xanthomonas campestris pv</i>. <i>vesicatoria</i> (lane 7–12) or <i>Ralstonia solanacearum</i> (lanes 13–18) at anealling temperatures of 58, 61, 63, 66, 68, 69°C; lane L: 1 kb ladder; lane 19: non-template control.

<p>Multiplex polymerase chain reaction showing specificity of three primer pairs to amplify <i>Pseudomonas syringae</i> pv. <i>tomato</i> (lane 1–6), <i>Xanthomonas campestris pv</i>. <i>vesicatoria</i> (lane 7–12) or <i>Ralstonia solanacearum</i> (lanes 13–18) at anealling temperatures of 58, 61, 63, 66, 68, 69°C; lane L: 1 kb ladder; lane 19: non-template control.</p

Experimental procedure for acoustic measurements.

<p>The method consists of two steps: 1. End-point PCR and 2. PCR product immobilization. Before addition of the PCR reaction, neutravidin protein is adsorbed on a clean gold QCM crystal and biotinylated DNA is immobilized through biotin-neutravidin interactions. Biomolecules are not drawn in scale.</p

Pathogen population growth and disease symptoms in tomato plants grown to the 4–5 true leaf stage (BBCH 104–105) following artificial infection (inoculation) with A: <i>Pseudomonas syringae</i> pv <i>tomato (Pto)</i> suspension which was: I.

<p>Infiltrated at a 5 ×10⁵ cfu ml^-1 concentration inside the leaf apoplast using a syringe, developing a necrotic lesion 4 dpi <b>II</b>. Sprayed at a 10⁸ cfu ml^-1 concentration on both sides of the leaf, developing indistinct dark, round spots (specks) after entry through the stomata and epidermal wounds 7 dpi. The increase in <i>Pto</i> population size was determined in leaf discs (3 triplicated samples) and over a 17 day time course following treatments and infection. Bars represent means ± SE in log10 scale; <i>dpi</i>: days post inoculation. <b>B</b>. <i>Ralstonia solanacearum</i> suspension drenched to pots containing tomato plants in the 4-5^th true leaf stage. Early symptoms appear in the youngest leaves having a flabby appearance followed by wilting of the whole plant 7 days post inoculation (dpi) due to invasion of xylem vessels and collapse of the vascular system.</p

The Effect of Silver Nanoparticles Size, Produced Using Plant Extract from Arbutus unedo, on Their Antibacterial Efficacy

Silver nanoparticles (AgNPs) have been demonstrated to restrain bacterial growth, while maintaining minimal risk in development of bacterial resistance and human cell toxicity that conventional silver compounds exhibit. Several physical and chemical methods have been reported to synthesize AgNPs. However, these methods are expensive and involve heavy chemical reduction agents. An alternative approach to produce AgNPs in a cost-effective and environmentally friendly way employs a biological pathway using various plant extracts to reduce metal ions. The size control issue, and the stability of nanoparticles, remain some of the latest challenges in such methods. In this study, we used two different concentrations of fresh leaf extract of the plant Arbutus unedo (LEA) as a reducing and stabilizing agent to produce two size variations of AgNPs. UV-Vis spectroscopy, Dynamic Light Scattering, Transmission Electron Microscopy, and zeta potential were applied for the characterization of AgNPs. Both AgNP variations were evaluated for their antibacterial efficacy against the gram-negative species Escherichia coli and Pseudomonas aeruginosa, as well as the gram-positive species Bacillus subtilis and Staphylococcus epidermidis. Although significant differences have been achieved in the nanoparticles’ size by varying the plant extract concentration during synthesis, the antibacterial effect was almost the same

Comparison of the acoustic ratios measured for the 3 different pathogens and the negative control.

<p>Comparison of the acoustic ratios measured for the 3 different pathogens and the negative control.</p

Cell Wall Chitosaccharides Are Essential Components and Exposed Patterns of the Phytopathogenic Oomycete Aphanomyces euteiches▿

Chitin is an essential component of fungal cell walls, where it forms a crystalline scaffold, and chitooligosaccharides derived from it are signaling molecules recognized by the hosts of pathogenic fungi. Oomycetes are cellulosic fungus-like microorganisms which most often lack chitin in their cell walls. Here we present the first study of the cell wall of the oomycete Aphanomyces euteiches, a major parasite of legume plants. Biochemical analyses demonstrated the presence of ca. 10% N-acetyl-d-glucosamine (GlcNAc) in the cell wall. Further characterization of the GlcNAc-containing material revealed that it corresponds to noncrystalline chitosaccharides associated with glucans, rather than to chitin per se. Two putative chitin synthase (CHS) genes were identified by data mining of an A. euteiches expressed sequence tag collection and Southern blot analysis, and full-length cDNA sequences of both genes were obtained. Phylogeny analysis indicated that oomycete CHS diversification occurred before the divergence of the major oomycete lineages. Remarkably, lectin labeling showed that the Aphanomyces euteiches chitosaccharides are exposed at the cell wall surface, and study of the effect of the CHS inhibitor nikkomycin Z demonstrated that they are involved in cell wall function. These data open new perspectives for the development of antioomycete drugs and further studies of the molecular mechanisms involved in the recognition of pathogenic oomycetes by the host plants

Engineered Polyamine Catabolism Preinduces Tolerance of Tobacco to Bacteria and Oomycetes1[C][W][OA]

Polyamine oxidase (PAO) catalyzes the oxidative catabolism of spermidine and spermine, generating hydrogen peroxide. In wild-type tobacco (Nicotiana tabacum ‘Xanthi’) plants, infection by the compatible pathogen Pseudomonas syringae pv tabaci resulted in increased PAO gene and corresponding PAO enzyme activities; polyamine homeostasis was maintained by induction of the arginine decarboxylase pathway and spermine was excreted into the apoplast, where it was oxidized by the enhanced apoplastic PAO, resulting in higher hydrogen peroxide accumulation. Moreover, plants overexpressing PAO showed preinduced disease tolerance against the biotrophic bacterium P. syringae pv tabaci and the hemibiotrophic oomycete Phytophthora parasitica var nicotianae but not against the Cucumber mosaic virus. Furthermore, in transgenic PAO-overexpressing plants, systemic acquired resistance marker genes as well as a pronounced increase in the cell wall-based defense were found before inoculation. These results reveal that PAO is a nodal point in a specific apoplast-localized plant-pathogen interaction, which also signals parallel defense responses, thus preventing pathogen colonization. This strategy presents a novel approach for producing transgenic plants resistant to a broad spectrum of plant pathogens