Botrytis cinerea Infection of Grape Flowers: Light and Electron Microscopical Studies of Infection Sites

ABSTRACT Specific floral organs including the calyptra, stigma, and receptacle area of glasshouse-grown grapevines (Vitis vinifera cv. Cabernet Sauvignon) were inoculated with aqueous suspensions of Botrytis cinerea conidia, and the initial steps involved in colonization and infection of the host tissues were studied for several days postinoculation using light microscopy as well as scanning and transmission electron microscopy. Conidia germinated on all floral organs examined and became attached to the host surface within 48 h after inoculation. However, in all cases the vast majority of conidia accumulated in a channel-like gap between the ovary and the calyx that extended in a narrowing fashion into the flower interior where the ovary joined the receptacle. Very few conidial germ tubes were detected in the style following inoculation of the stigma, and no evidence for their growth toward the ovaries could be found. In contrast, hyphae were more abundant in the receptacle area, regardless of the site of inoculation. Tips of the calyx became necrotic and mycelium formed in the gap between ovary and calyx within 72 h following inoculation, providing a major point of colonization and infection. B. cinerea colonized dehiscent calyptras within 72 h of inoculation, providing a potential source of inoculum from calyptras that remained stuck in the cluster. The results suggest that the grape flower's receptacle area is the predominant site of infection for B. cinerea, although a minor portion of infections may also occur through the stigma and style.     

A trend test for the analysis of multiple paternity

The aquatic warbler, Acrocephalus paludicola, is one of a few species in which nestlings from a single nest can be sired by up to four different fathers. Data from a DNA fingerprinting study suggest that there is a trend: the larger the number of fathers in a brood the larger is the number of nestlings. However, the number of young within a single nest cannot be smaller than the number of fathers. This restriction causes an inherent trend and, consequently, can lead to a false-positive trend test result. For this nonstandard situation, we propose a trend test that differentiates between the inherent trend and a “real” trend, that is, a larger brood size through multiple paternity. Using DNA fingerprinting data, we performed a randomization test using the proposed new trend test statistic and obtained a significant result (P=0.047). This indicates that a larger number of fathers per brood is associated with a larger brood size. In addition, we consider an umbrella alternative, that is, a downturn in effect may occur after the optimal number of fathers per brood is exceeded. An appropriate test for this alternative also leads to a significance (P=0.011).     

A data-driven fuzzy approach to simulate the critical shear stress of mixed cohesive/non-cohesive sediments

Purpose

The critical shear stress of cohesive and mixed cohesive/non-cohesive sediments is affected by multiple interacting physical, chemical and biological parameters. There are various mathematical approaches in the scientific literature for computing critical shear stress. However, processes that influence sediment stability are still not fully understood, and available formulas differ considerably. These discrepancies in the literature arise from random system behaviour (natural variability of the sediments), different definitions of the critical shear stress, different measurement techniques and different model frameworks (scope of the parameters, undisturbed versus artificial sediment samples). While analytical approaches fail to address the involved uncertainties, fuzzy logic-based models integrate uncertainty and imprecision.

Materials and methods

With this in mind, a data-driven neuro-fuzzy model (ANFIS) was used to determine the critical shear stress based on sediment characteristics such as wet bulk density and grain size distribution. In order to select model predictors systematically, an automated stepwise regression algorithm was applied. The database for this analysis consisted of 447 measurements of the critical shear stress originating from 64 undisturbed sediment samples.

Results and discussion

The study identified clay content as the primarily controlling variable for erosion resistance. Depending on the characteristics of the sampling location, the bulk density was also selected as a model predictor. In comparison to analytical models that are available in the scientific literature, the fuzzy model achieved higher correlation coefficients between measured and predicted data.

Conclusions

The neuro-fuzzy-model includes uncertainties of input variables and their interactions directly. Thus, it provides a reliable method for the prediction of erosion thresholds of cohesive/non-cohesive mixtures. It was also shown that this approach requires fewer measured variables as well as fewer assumptions than the models it was compared to.

     

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing analytical validation

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomic evaluations, compositional analyses, animal nutrition, and classical toxicology evaluations. Because analytical technologies are rapidly developing, proteome analysis based on two-dimensional gel electrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DE method was established for the qualitative and quantitative analysis of the seed proteome of Arabidopsis thaliana with the following validation parameters examined: (1) source and scope of variation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolves proteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and is sensitive enough to detect protein levels in the low nanogram range. The separation of the proteins was demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI and MM directions, respectively. The mean coefficient of variation of 254 matched spot qualities was found to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R2 > 0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. Therefore, this method could be used to interrogate proteome alterations such as a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point, and/or quantity of a protein.     

A combined hydraulic and toxicological approach to assess re-suspended sediments during simulated flood events—part II: an interdisciplinary experimental methodology

Purpose

Flood events are expected to increase both in intensity and frequency due to climate change in the near future. From an environmental toxicology perspective, there is concern that such flood events could lead to the remobilization of contaminated sediment layers in rivers. The aim of this pilot study was to establish a novel and interdisciplinary framework combining methods of hydrodynamic engineering and ecotoxicological assessment to enable investigation of the potential risks associated with such remobilization events.

Materials and methods

Formulated sediment was prepared according to OECD guideline 218 and spiked with a mixture of four polycyclic aromatic hydrocarbons (phenanthrene, chrysene, pyrene, benzo[a]pyrene) at concentrations of 3.3?C8.3?mg?kg^?1 dry weight. Rainbow trout (Oncorhynchus mykiss) were exposed as test animals to re-suspended sediments in three out of five experiments. The experiments were carried out in an annular flume designed to investigate transport behaviour of fine-grained sediments. Several physicochemical (e.g. pH) and sedimentological (e.g. turbidity) parameters were measured to characterise environmental conditions and erosion behaviour of sediments. Furthermore, exposure concentrations were measured by means of an in vitro assay (7-ethoxyresorufin-o-deethylase (EROD), RTL-W1 cell assay) and chemical analysis.

Results and discussion

Preparation and spiking of large amounts of formulated sediments were feasible but not practical. Successful spiking could be confirmed by the bioanalytical methods with the spiked sediments showing significantly elevated EROD induction compared to control sediments. Conditions within the annular flume remained stable throughout all experiments and were adequate to support rainbow trout. Flood events were successfully simulated, resulting in re-suspension of formulated sediment. Different erosion behaviours of sediments during the simulated flood events were observed and could be associated with changes in microbial composition of sediments due to differences in storage conditions. Therefore, maintaining constant storage conditions of formulated sediments is crucial to enable consistency and comparability among erosion experiments.

Conclusions

This study clearly demonstrated the feasibility of a combined hydro-toxicological approach in support of the investigation of the potential ecotoxicological relevance of sediment re-suspension events. However, based on the results presented here, it is recommended to include additional physicochemical parameters, such as redox potential and conductivity, and to extend the experimental setup to natural sediments and different aquatic organisms. Future studies will use natural sediments containing representative microbial communities and extracellular polymeric substances to enable extrapolation from the annular flume to conditions in natural flowing waters.     

Physical properties of a Luvisol for different long‐term fertilization treatments: I. Mesoscale capacity and intensity parameters

The physical properties of a Luvisol derived from loess near Bonn, Germany, under different long‐term fertilization treatments were examined. For the investigation of the impact of farmyard manure (FYM) on soil strength at the mesoscale (100 to 300 cm³ soil cores), undisturbed samples were taken from two different depths (10 and 40 cm), either with no fertilization at all, with full mineral fertilization, with FYM only, and with both mineral and organic fertilization. We investigated hydraulic and mechanical parameters, namely precompression stress, pore‐size distribution, saturated hydraulic and air conductivity, and calculated pore connectivity. Long‐term organic fertilization resulted in significantly more and coarser pores which in addition were more conductant and mechanically stronger by trend. Mineral fertilization also increased pore volume by trend but not pore functionality. Mechanical strength generally increased with fertilization by trend, however, was reduced again when organic and mineral fertilization were combined. Nonetheless, FYM led to relatively higher soil strength as the FYM‐treated plots with lower bulk density attained similar soil strength as the unfertilized but denser plots and thus supported the soil‐improving impact of organic amendments. The subsoil physical properties were rather unaffected by fertilization, but were dominated by texture.     

Fluxes,pools, and turnover of mercury in Swedish forest lakes

In boreal forest lakes, high Hg concentrations in fish are common, even in remote areas. In this paper, the effects of atmospheric Hg pollution in Sweden are synthesized and related to a concept based on the strong interaction of Hg with biogenic matter (Hg/B). Based on this concept, a compartment model is developed to predict concentrations, pool sizes, flux rates and turnover times of Hg along the biogeochemical cycle, including atmosphere, forest soils, surface runoff, lake waters, and aquatic biota. The aim is to provide a conceptual framework, both for a comprehensive mechanistic model, and for predictions from readily available information, such as regional data on acid deposition, air temperature and surface runoff, and local data on the trophic status of lakes with respect to humus and nutrient concentrations. The model is in good agreement with observations from recent Swedish field studies in all compartments. It suggests a strong influence of climate on the susceptibility of soil and lake ecosystems in the boreal region to Hg contamination. The high Hg concentrations in fish from forest lakes can be largely attributed to the low productivity of both terrestrial and aquatic biota. The impact of historical point sources of Hg is considered, as well as the slow turnover of Hg in forest soils, both resulting in elevated fish Hg levels in humic lakes for centuries following atmospheric deposition.     

Short‐term degradation of semiarid grasslands—results from a controlled‐grazing experiment in Northern China

The assessment of grassland degradation due to overgrazing is a global challenge in semiarid environments. In particular, investigations of beginning steppe degradation after a change or intensification of the land use are needed in order to detect and adjust detrimental land‐use management rapidly and thus prevent severe damages in these sensitive ecosystems. A controlled‐grazing experiment was established in Inner Mongolia (China) in 2005 that included ungrazed (UG) and heavily grazed plots with grazing intensities of 4.5 (HG4.5) and 7.5 (HG7.5) sheep per hectare. Several soil and vegetation parameters were investigated at all sites before the start of the experiment. Topsoil samples were analyzed for soil organic C (SOC), total N (N_tot), total S (S_tot), and bulk density (BD). As vegetation parameters, aboveground net primary productivity (ANPP), tiller density (TD), and leaf‐area index (LAI) were determined. After 3 y of the grazing experiment, BD increased and SOC, N_tot, S_tot, ANPP, and LAI significantly decreased with increasing grazing intensity. These sensitive parameters can be regarded as early‐warning indicators for degradation of semiarid grasslands. Vegetation parameters were, however, more sensitive not only to grazing but also to temporal variation of precipitation between 2006 and 2008. Contrary, soil parameters were primarily affected by grazing and resistant against climatic variations. The assessment of starting conditions in the study area and the application of defined grazing intensities is essential for the investigation of short‐term degradation in semiarid environments.     

Reconciling 14C and minirhizotron‐based estimates of fine‐root turnover with survival functions

The turnover of fine‐roots is a crucial component for the input of C to the soil. The amount of root litter input is depending on estimates of turnover times from different techniques. Turnover times from fine‐root cameras (minirhizotrons) often yield 75% higher root litter input estimates than turnover times estimated with the bomb‐radiocarbon signature of fine roots. We introduce a generic framework for the analysis of fine‐root ¹⁴C with different survival functions. So far, mostly an exponential function has been used to estimate the turnover time and mean age of fine roots. In the context of the introduced survival function framework we clarify the terms turnover time, mean residence time, mean longevity, and mean age, which are commonly used in studies of root turnover. Using a unique time series of fine‐root ¹⁴C (Fröberg 2012), we test if survival functions other than the exponential function are better in accordance with turnover‐time estimates commonly found with other methods. A survival function that corresponds to a two‐pool model was best in agreement with minirhizotron‐based estimates (mean residence time of 1.9 y). We argue that using fine‐root ¹⁴C and minirhizotron time‐to‐death data together would give the best constraints on fine‐root turnover. At the same time this could allow quantifying systematic biases inherent to both techniques.