Physical Modes of Action of Fungicides Against Apple Scab: Timing Is Everything, but Dose Matters.

The efficacy of currently available fungicides against apple scab, caused by the fungal pathogen Venturia inaequalis, was investigated in relation to when growers spray (ahead, during, or after rain) and how the spray reaches the target. The adaxial surface of individual leaves of potted trees were sprayed and then inoculated with ascospores of V. inaequalis, to establish dose-response curves for each fungicide. Discriminatory doses providing 50 and 90% symptom inhibition (EC50 and EC90, respectively) in sprays mimicking applications ahead of rain were used for experiments imitating alternative spray timings. Sprays were either applied during the spore germination phase or early or late after infection onset (either 336 or 672 degree-hours after inoculation, respectively), corresponding to grower spray schedules. Experiments were also carried out with sprays applied on the abaxial leaf surface to investigate fungicide efficacy through the leaf lamina. For all fungicides, the best efficacy was observed when sprays were applied during germination, followed by applications ahead of inoculation. Some products maintained equal or better efficacy at early infection, while efficacy in late infection dropped for all products, clearly indicating that this spray timing should be avoided. Some products with postinfection efficacy also showed translaminar efficacy. The close relationship found between EC50 of the active ingredients on potted trees and the label rate could help improve spraying decisions and reduce costs.

Forensic application of position-specific isotopic analysis of trinitrotoluene (TNT) by NMR to determine 13C and 15N intramolecular isotopic profiles.

2,4,6-trinitrotoluene (TNT) is a molecule which is easily identified with current instrumental techniques but it is generally impossible to distinguish between sources of the same substance (TNT). To overcome this difficulty, we present a multi stable isotope approach using isotope ratio monitoring by mass spectrometry (irm-MS) and Nuclear Magnetic Resonance spectrometry (irm-NMR). In the one hand, irm-MS provides bulk isotopic composition at natural abundance in 13C and 15N. The range of variation between samples is rather small particularly for 13C. In the other hand, irm-13C NMR and irm-15N NMR enable the determination of positional intramolecular 13C/12C ratios (δ13Ci) and 15N/14N ratios (δ15Ni) with high precision that lead to larger variation between samples. The present work reports an application of the recent methodology using irm-15N NMR to determine position-specific 15N isotope content of TNT. The interest of this methodology is compared to irm-13C NMR and irm-MS (13C and 15N) in terms of TNT samples discrimination. Thanks to the use of irm-NMR the results show a unique isotopic fingerprint for each TNT which enable origin discrimination between the samples without ambiguity.

Isotopomics by isotope ratio monitoring by 13 C nuclear magnetic resonance spectrometry on cutting agents in heroin: A new approach for illicit drugs trafficking route elucidation.

In the battle against the illicit drugs market, methodologies have been developed by forensic laboratories to address the determination of the origin and dismantlement of the trafficking route for various target molecules such as heroin and cocaine. These drug profiling methods are not straightforward, especially when the target molecules are synthetic and very pure, resulting in poorly informative impurity profiles, e.g. new psychoactive substances and cutting agents. A tool based on the determination of intramolecular isotopic profiles has been developed to provide origin discrimination with a new way to profile seized cutting agents and heroin samples. Whereas stable isotope analyses by mass spectrometry give the bulk isotopic composition, nuclear magnetic resonance gives direct access to the position-specific isotope content at natural abundance. This report shows how both 13 C NMR spectrometry and 13 C, 15 N MS might provide complementary and valuable information to link seized caffeine and paracetamol to their origin. Here, isotopic ratio monitoring by 13 C NMR (irm-13 C NMR) offers additional benefits over irm-MS in its capability to determine a detailed isotopic profile, leading to a better method to distinguish different caffeine and paracetamol batches.

Distribution of the Infection Time of Ascospores of Venturia inaequalis.

In many areas where spring is wet, fungicides are applied in relation to rain events that trigger ejection of ascospores of Venturia inaequalis, which cause primary infections of apple scab. Past studies established the rate of ejection during rain in relation to light and temperature, and determined the wetting time required for infection. Simulation software uses this information to calculate risk and help time sprays accordingly. However, the distribution of the infection time required by a population of spores landed on leaves was never studied, and assumptions were used. To estimate this, we inoculated ascospores of V. inaequalis on potted trees at different temperatures for specific wetting times. Lesions were enumerated after incubation. Lesions increased with wetness time and leveled off once the slowest spores infected the host, closely matching the monomolecular model. Wetness hours were best adjusted for temperature using the Yin equation. The minimum infection time on the youngest leaves was about 5 h, matching results from previous studies, whereas half the lesions appeared after 7 h of infection. Infection times for leaves with ontogenic resistance were longer. Our results improve current software estimates and may improve spraying decisions.

Impact of Exclusion Netting Row Covers on 'Honeycrisp' Apple Trees Grown under Northeastern North American Conditions: Effects on Photosynthesis and Fruit Quality.

Exclusion nets have been used successfully to protect fruit from insect pests of apples under various conditions, but the effect of each particular netting system on the plant itself has rarely been investigated. In this study, a complete exclusion system-in which the soil is also excluded-was used to grow 'Honeycrisp' apples for six years in southern Quebec, Canada. Abiotic conditions, as well as plant photosynthesis and fruit quality characteristics (colour, firmness, size, sugar content, number of seeds, ripeness and skin integrity) and yield were estimated yearly and compared in netted (either with or without a rainproof top) and unnetted row units. Although annual variations were high and results showed little or no difference between netted and unnetted rows for all measured variables, with the following exceptions; colour (increased red surface on fruits from unnetted rows some years), size (fruits from unnetted rows were smaller) and maturity (fruits from unnetted rows matured slightly faster). Fruits produced under nets had fewer microcracks at the surface than fruits produced without nets. Reduced cracking possibly helped decrease sooty blotch and flyspeck incidence and severity. Impacts for pest control and prospects for pesticide-free production are discussed.

Position-specific 15 N isotope analysis in organic molecules: A high-precision 15 N NMR method to determine the intramolecular 15 N isotope composition and fractionation at natural abundance.

The position-specific 15 N isotope content in organic molecules, at natural abundance, is for the first time determined by using a quantitative methodology based on 15 N Nuclear Magnetic Resonance (NMR) spectrometry. 15 N NMR spectra are obtained by using an adiabatic "Full-Spectrum" INEPT sequence in order to make possible 15 N NMR experiments with a high signal-to-noise ratio (>500), to reach a precision with a standard deviation below 1‰ (0.1%). This level of precision is required for observing small changes in 15 N content associated to 15 N isotope effects. As an illustration, the measurement of an isotopic enrichment factor ε for each 15 N isotopomer is presented for 1-methylimidazole induced during a separation process on a silica column. The precision expressed as the long-term repeatability of the methodology is good enough to evaluate small changes in the 15 N isotope contents for a given isotopomer. As observed for 13 C, inverse and normal 15 N isotope effects occur concomitantly, giving access to new information on the origin of the 15 N isotope effects, not detectable by other techniques such as isotope ratio measured by Mass Spectrometry for which bulk (average) values are obtained.

Full Spectrum Isotopic 13C NMR Using Polarization Transfer for Position-Specific Isotope Analysis.

For the last ten years, quantitative isotope ratio monitoring 13C NMR (irm-13C NMR) has been successfully tested and proven as an efficient tool for the determination of position-specific 13C/12C ratios. Several applications in different domains have shown the interest in this technique. In the context of origin assignment, the possibility to track the distribution network of illicit drugs or cutting agents is of prime importance. However irm-13C NMR still suffers from a relative lack of sensitivity limiting its dissemination among control laboratories. Improvements were proposed to reduce experiment time by using the INEPT sequence ("Insensitive Nuclei Enhanced by Polarization Transfer") based on polarization transfer from highly sensitive 1H to less sensitive 13C. Several applications based on the use of the one bond scalar coupling between 1H and 13C (1 JCH) have shown the potential of this methodology in terms of short experimental duration. However, the isotopic information given by quaternary carbons was lost. The aim of this study is to extend this approach by using short- and long-range coupling (1 JCH, 2 JCH, and 3 JCH) in order to have access to all 13C/12C position-specific ratios, i.e., acquisition of the full spectrum (FS-INEPT). It is shown that this innovative tool provides both sensitivity gain-thanks to the long-range polarization transfer-and appropriate repeatability. The relative isotopic profiles allowed the classification of two cutting agents, caffeine and paracetamol (acetaminophen), according to their origin, as it was previously observed with "classical" irm-13C NMR but consuming much less sample and/or reducing the experimental time.

The new face of isotopic NMR at natural abundance.

The most widely used method for isotope analysis at natural abundance is isotope ratio monitoring by Mass Spectrometry (irm-MS) which provides bulk isotopic composition in 2 H, 13 C, 15 N, 18 O or 34 S. However, in the 1980s, the direct access to Site-specific Natural Isotope Fractionation by Nuclear Magnetic Resonance (SNIF-NMRTM ) was immediately recognized as a powerful technique to authenticate the origin of natural or synthetic products. The initial - and still most popular - application consisted in detecting the chaptalization of wines by irm-2 H NMR. The approach has been extended to a wide range of methodologies over the last decade, paving the way to a wide range of applications, not only in the field of authentication but also to study metabolism. In particular, the emerging irm-13 C NMR approach delivers direct access to position-specific 13 C isotope content at natural abundance. After highlighting the application scope of irm-NMR (2 H and 13 C), this article describes the major improvements which made possible to reach the required accuracy of 1‰ (0.1%) in irm-13 C NMR. The last part of the manuscript summarizes the different steps to perform isotope analysis as a function of the sample properties (concentration, peak overlap) and the kind of targeted isotopic information (authentication, affiliation). Copyright © 2016 John Wiley & Sons, Ltd.