The effects of surface structure mutations in <i>Arabidopsis thaliana</i> on the polarization of reflections from virus-infected leaves

<div><p>The way in which light is polarized when reflected from leaves can be affected by infection with plant viruses. This has the potential to influence viral transmission by insect vectors due to altered visual attractiveness of infected plants. The optical and topological properties of cuticular waxes and trichomes are important determinants of how light is polarized upon reflection. Changes in expression of genes involved in the formation of surface structures have also been reported following viral infection. This paper investigates the role of altered surface structures in virus-induced changes to polarization reflection from leaves. The percentage polarization of reflections from <i>Arabidopsis thaliana cer5</i>, <i>cer6</i> and <i>cer8</i> wax synthesis mutants, and the <i>gl1</i> leaf hair mutant, was compared to those from wild-type (WT) leaves. The <i>cer5</i> mutant leaves were less polarizing than WT on the adaxial and abaxial surfaces; <i>gl1</i> leaves were more polarizing than WT on the adaxial surfaces. The <i>cer6</i> and <i>cer8</i> mutations did not significantly affect polarization reflection. The impacts of <i>Turnip vein clearing virus</i> (TVCV) infection on the polarization of reflected light were significantly affected by <i>cer5</i> mutation, with the reflections from <i>cer5</i> mutants being higher than those from WT leaves, suggesting that changes in <i>CER5</i> expression following infection could influence the polarization of the reflections. There was, however, no significant effect of the <i>gl1</i> mutation on polarization following TVCV infection. The <i>cer5</i> and <i>gl1</i> mutations did not affect the changes in polarization following <i>Cucumber mosaic virus</i> (CMV) infection. The accumulation of TVCV and CMV did not differ significantly between mutant and WT leaves, suggesting that altered expression of surface structure genes does not significantly affect viral titres, raising the possibility that if such regulatory changes have any adaptive value it may possibly be through impacts on viral transmission.</p></div

Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus , is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the Msp I satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus , up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation

Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests.

High-throughput sequencing (HTS) technologies have the potential to become one of the most signi cant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, con rming and interpreting results. These guidelines cover any HTS test used for the detection and identi cation of any plant pest (viroid, virus, bacteria, phytoplasma, fungi and fungus-like protists, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health

Generating and testing ecological hypotheses at the pondscape with environmental DNA metabarcoding: A case study on a threatened amphibian

Background: Environmental DNA (eDNA) metabarcoding is revolutionizing biodiversity monitoring, but has unrealized potential for ecological hypothesis generation and testing. Aims: Here, we validate this potential in a large-scale analysis of vertebrate community data generated by eDNA metabarcoding of 532 UK ponds. Materials & Methods: We test biotic associations between the threatened great crested newt (Triturus cristatus) and other vertebrates as well as abiotic factors influencing T.cristatus detection at the pondscape. Furthermore, we test the status of T.cristatus as an umbrella species for pond conservation by assessing whether vertebrate species richness is greater in ponds with T.cristatus and higher T.cristatus Habitat Suitability Index (HSI) scores. Results: Triturus cristatus detection was positively correlated with amphibian and waterfowl species richness. Specifically, T.cristatus was positively associated with smooth newt (Lissotriton vulgaris), common coot (Fulica atra), and common moorhen (Gallinula chloropus), but negatively associated with common toad (Bufo bufo). Triturus cristatus detection did not significantly decrease as fish species richness increased, but negative associations with common carp (Cyprinus carpio), three-spined stickleback (Gasterosteus aculeatus), and ninespine stickleback (Pungitius pungitius) were identified. Triturus cristatus detection was negatively correlated with mammal species richness, and T.cristatus was negatively associated with gray squirrel (Sciurus carolinensis). Triturus cristatus detection was negatively correlated with larger pond area, presence of inflow, and higher percentage of shading, but positively correlated with HSI score, supporting its application to T.cristatus survey. Vertebrate species richness was significantly higher in T.cristatus ponds and broadly increased as T.cristatus HSI scores increased. Discussion: We reaffirm reported associations (e.g., T.cristatus preference for smaller ponds) but also provide novel insights, including a negative effect of pond inflow on T.cristatus. Conclusion: Our findings demonstrate the prospects of eDNA metabarcoding for ecological hypothesis generation and testing at landscape scale, and dramatic enhancement of freshwater conservation, management, monitoring, and research