Quantification of the total suspended matter concentration in the sea breaking zone from in situ measurements and remotely sensed data - two empirical approaches

Remote sensing techniques can be used to calculate suspended sediment concentrations and to understand the flux and distribution of sediments driven by mechanisms such as tides and waves, river discharges, etc. The main objective of this study is the quantification of the Total Suspended Matter (TSM) concentration in the sea breaking zone for a particular area of the Portuguese coast, around Aveiro. The methodology used was based on in situ measurements and multi spectral satellite images. In situ experimental techniques (maritime platform, aerial platform, simulation on the beach and water sample collection in the breaking zone) were used to determine a relationship between the TSM concentration and the seawater reflectance in the breaking zone. Spectral reflectance was measured with a spectroradiometer and water samples were simultaneously collected. Empirical relationships were established between TSM concentration and the equivalent reflectance values for sensors SPOT/HRVIR, TERRA/ASTER and Landsat/TM at visible and Near Infra Red (NIR) bands computed from the experimental data. Satellite images from ASTER, SPOT HRVIR and Landsat TM were used together with the same empirical models. These satellite images were calibrated and atmospherically corrected. Equations of linear, polynomial, logarithmic, power and exponential models were tested for the satellite image bands on the visible and near infrared. The coefficients of determination (R-2) were also calculated for each model. The results obtained from the two approaches, in situ measurements and directly from the multi spectral satellite images, were analysed

Evolutionary and Experimental Assessment of Novel Markers for Detection of Xanthomonas euvesicatoria in Plant Samples

BACKGROUND: Bacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens. METHODOLOGY: In this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants. SIGNIFICANCE: This study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics

Quantification of the total suspended matter concentration in the sea breaking zone from in situ measurements and remotely sensed data - two empirical approaches

Remote sensing techniques can be used to calculate suspended sediment concentrations and to understand the flux and distribution of sediments driven by mechanisms such as tides and waves, river discharges, etc. The main objective of this study is the quantification of the Total Suspended Matter (TSM) concentration in the sea breaking zone for a particular area of the Portuguese coast, around Aveiro. The methodology used was based on in situ measurements and multi spectral satellite images. In situ experimental techniques (maritime platform, aerial platform, simulation on the beach and water sample collection in the breaking zone) were used to determine a relationship between the TSM concentration and the seawater reflectance in the breaking zone. Spectral reflectance was measured with a spectroradiometer and water samples were simultaneously collected. Empirical relationships were established between TSM concentration and the equivalent reflectance values for sensors SPOT/HRVIR, TERRA/ASTER and Landsat/TM at visible and Near Infra Red (NIR) bands computed from the experimental data. Satellite images from ASTER, SPOT HRVIR and Landsat TM were used together with the same empirical models. These satellite images were calibrated and atmospherically corrected. Equations of linear, polynomial, logarithmic, power and exponential models were tested for the satellite image bands on the visible and near infrared. The coefficients of determination (R-2) were also calculated for each model. The results obtained from the two approaches, in situ measurements and directly from the multi spectral satellite images, were analysed

PCR validation.

<p>The selected primer-pairs were tested for efficiency using eight different <i>Xeu</i> strains. For each assay, three different annealing temperatures were tested: 57°C, 59°C and 61°C.</p

Detection of BSX in infected plant material using an inverted dot blot platform.

<p>Crude bacterial suspensions, obtained from tomato and pepper plants leaves after one and two weeks of infection with <i>Xeu</i> 905, were used as templates for PCR enrichment using the markers' primer pairs. PCR products corresponding to each plant were labeled with Digoxigenin and used as probes. Purified DNA from <i>Xeu</i> 905 was used as positive control. Negative controls consisted of tomato plants infected with <i>Pst</i> DC3000 for 2 weeks and uninfected plants. The raw ChemiDoc captures and processed images, using the automatic image analysis algorithm, are shown.</p

Neighbor-Joining Tree based on the concatenated sequences of four housekeeping genes of several <i>Xanthomonas</i>.

<p>The sequences of the housekeeping genes <i>atpD</i>, <i>dnaK</i>, <i>efp</i> and <i>gyrB</i> were concatenated and used to infer the MLST profile of <i>X. euvesicatoria</i> and <i>X. vesicatoria</i> strains used in this study, which are highlighted in yellow. The Neighbor-Joining tree was derived from the TN93+G+I model and a bootstrap analysis of 1000 replicates.</p

Dot blot validation of selected probes.

<p>Nine probes were evaluated with total DNA from a collection of BSX, consisting of 19 <i>Xeu</i>, five <i>Xv</i>, three <i>Xg</i> and two <i>Xp</i> strains. Probability values, obtained with a customized MATLAB algorithm for the automatic data analysis, are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037836#pone-0037836-t003" target="_blank">Table 3</a>.</p

List of bacterial strains used in this study.

*<p>LMG-Belgian Co-Ordinated collections of micro-organisms, Gent, Belgium; CPBF-Colecção Portuguesa de Bactérias Fitopatogénicas, Lisboa, Portugal; NCPPB-National Collection of Plant Pathogenic Bacteria, York, United Kingdom.</p>a<p>- Bacterial spot-causing xanthomonads (BSX);</p>b<p>- Pathovar reference strain;</p>c<p>- Type strain; NM- Not mentioned.</p

Detection of BSX in infected plant material using a duplex PCR (markers XV7 and XV11).

<p>Tomato and pepper plants inoculated with <i>Xeu</i> 905, <i>Xv</i> 919, <i>Xg</i> 962 and <i>Xp</i> 4321were processed after one and two weeks to obtain crude bacterial suspensions used as PCR templates. Plants inoculated with <i>Pst</i> DC3000 were used as controls. M – DNA marker (GeneRuler DNA Ladder Mix); Ø-Duplex PCR using distilled water as template; C- healthy tomato and pepper plants.</p

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field