Colonization of Vitis vinifera L. by the Endophyte Trichoderma sp. Strain T154: Biocontrol Activity Against Phaeoacremonium minimum

[EN] Trichoderma strains used in biological control products usually exhibit high efficiency in the control of plant diseases. However, their behavior under field conditions is difficult to predict. In addition, the potential of indigenous strains has been poorly assayed as well as their possible behavior as endophytes. Hence, niche colonization is a key feature for an effective protection. In this study, we aimed to: (i) explore the possibility of using a new Trichoderma strain isolated from vine to control pathogens, (ii) study the in planta interaction with the pathogen Phaeoacremonium minimum W. Gams, Crous, M.J. Wingf. & L. Mugnai (formerly Phaeoacremonium aleophilum), a pioneer fungus involved in Grapevine Trunk Diseases (GTDs) such as esca. For this purpose, fluorescently tagged Trichoderma sp. T154 and a P. minimum strain were used for scanning electron microscopy and confocal scanning laser microscopy analyses. Data showed that the Trichoderma strain is able to colonize plants up to 12 weeks post inoculation and is located in xylem, fibers, as well as in parenchymatic tissues inside the wood. The beneficial fungus reduced colonization of the esca-related pathogen colonizing the same niches. The main observed mechanism involved in biocontrol of Trichoderma against the esca pathogen was spore adhesion, niche exclusion and only few typical hypha coiling was found between Trichoderma and the pathogen. These results suggest that the Trichoderma strain has potential for reducing the colonization of Phaeoacremonium minimum and thus, an inoculation of this biological control agent can protect the plant by limiting the development of GTD, and the strain can behave as an endophyte.SIThe grant awarded to GC-H (FPU15/04681) comes from the Ministry of Education, Culture, and Sport (Spain). We thank Pago de Carraovejas winery for the project “GLOBALVITI IDI- 20120746” “Solució n global para mejorar la producció n vitivinı́ cola frente al cambio climá tico basada en robó tica, tecnologı́ a IT y en estrategias biotecnoló gicas y de manejo del viñedo” (Global solution for enhancing viticulture production against: climatic change based on: robotics, IT technology, biotechnological strategies, and vineyard management) that was granted by the Centro para el Desarrollo Tecnoló gico Industrial –CDTI-. SC and MG received funding via DaFNE Project Nr. 101384 from the Austrian Federal Ministry for Sustainability and Tourism (BMNT)

Amplitude and frequency of wetting and drying cycles drive N2_{2} and N2_{2}O emissions from a subtropical pasture

This study investigated the effects of irrigation frequency on N$_{2}$ and N$_{2}$O emissions from an intensively managed pasture in the subtropics. Irrigation volumes were estimated to replace evapotranspiration and were applied either once (low frequency) or split into four applications (high frequency). To test for legacy effects, a large rainfall event was simulated at the end of the experiment. Over 15 days, 7.9 ± 2.7 kg N$_{2}$ + N$_{2}$O-N ha$^{-1}$ was emitted on average regardless of irrigation frequency, with N$_{2}$O accounting for 25% of overall N$_{2}$ + N$_{2}$O. Repeated, small amounts of irrigation produced an equal amount of N$_{2}$ + N$_{2}$O losses as a single, large irrigation event. The increase in N$_{2}$O emissions after the large rainfall event was smaller in the high-frequency treatment, shifting the N$_{2}$O/(N$_{2}$O + N$_{2}$) ratio towards N$_{2}$, indicating a treatment legacy effect. Cumulative losses of N$_{2}$O and N$_{2}$ did not differ between treatments, but higher CO$_{2}$ emissions were observed in the high-frequency treatment. Our results suggest that the increase in microbial activity and related O$_{2}$ consumption in response to small and repeated wetting events can offset the effects of increased soil gas diffusivity on denitrification, explaining the lack of treatment effect on cumulative N$_{2}$O and N$_{2}$ emissions and the abundance of N cycling marker genes. The observed legacy effect may be linked to increased mineralisation and subsequent increased dissolved organic carbon availability, suggesting that increased irrigation frequency can reduce the environmental impact (N$_{2}$O), but not overall magnitude of N$_{2}$O and N$_{2}$ emissions from intensively managed pastures

Gross Ammonification and Nitrification Rates in Soil Amended with Natural and NH4-Enriched Chabazite Zeolite and Nitrification Inhibitor DMPP

Using zeolite-rich tuffs for improving soil properties and crop N-use efficiency is becoming popular. However, the mechanistic understanding of their influence on soil N-processes is still poor. This paper aims to shed new light on how natural and NH4+-enriched chabazite zeolites alter short-term N-ammonification and nitrification rates with and without the use of nitrification inhibitor (DMPP). We employed the 15N pool dilution technique to determine short-term gross rates of ammonification and nitrification in a silty-clay soil amended with two typologies of chabazite-rich tuff: (1) at natural state and (2) enriched with NH4+-N from an animal slurry. Archaeal and bacterial amoA, nirS and nosZ genes, N2O-N and CO2-C emissions were also evaluated. The results showed modest short-term effects of chabazite at natural state only on nitrate production rates, which was slightly delayed compared to the unamended soil. On the other hand, the addition of NH4+-enriched chabazite stimulated NH4+-N production, N2O-N emissions, but reduced NO3-N production and abundance of nirS-nosZ genes. DMPP efficiency in reducing nitrification rates was dependent on N addition but not affected by the two typologies of zeolites tested. The outcomes of this study indicated the good compatibility of both natural and NH4+-enriched chabazite zeolite with DMPP. In particular, the application of NH4 +-enriched zeolites with DMPP is recommended to mitigate short-term N losses

Effect of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on N-turnover, the N2_{2}O reductase-gene nosZ and N2_{2}O:N2_{2} partitioning from agricultural soils

Nitrification inhibitors (NIs) have been shown to reduce emissions of the greenhouse gas nitrous oxide (N$_{2}$O) from agricultural soils. However, their N$_{2}$O reduction efficacy varies widely across different agro-ecosystems, and underlying mechanisms remain poorly understood. To investigate effects of the NI 3,4-dimethylpyrazole-phosphate (DMPP) on N-turnover from a pasture and a horticultural soil, we combined the quantification of N$_{2}$ and N$_{2}$O emissions with $^{15}$N tracing analysis and the quantification of the N$_{2}$O-reductase gene (nosZ) in a soil microcosm study. Nitrogen fertilization suppressed nosZ abundance in both soils, showing that high nitrate availability and the preferential reduction of nitrate over N$_{2}$O is responsible for large pulses of N$_{2}$O after the fertilization of agricultural soils. DMPP attenuated this effect only in the horticultural soil, reducing nitrification while increasing nosZ abundance. DMPP reduced N$_{2}$O emissions from the horticultural soil by >50% but did not affect overall N$_{2}$ + N$_{2}$O losses, demonstrating the shift in the N$_{2}$O:N$_{2}$ ratio towards N$_{2}$ as a key mechanism of N$_{2}$O mitigation by NIs. Under non-limiting NO$_{3}$$^{-}$ availability, the efficacy of NIs to mitigate N$_{2}$O emissions therefore depends on their ability to reduce the suppression of the N$_{2}$O reductase by high NO$_{3}$$^{-}$ concentrations in the soil, enabling complete denitrification to N$_{2}$

A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions

Traditional chromatin analysis methods only test one locus at the time or use different templates for each locus, making a standardized analysis of large genomic regions or many co-regulated genes at different loci a difficult task. On the other hand, genome-wide high-resolution mapping of chromatin accessibility employing massive parallel sequencing platforms generates an extensive data set laborious to analyse and is a cost-intensive method, only applicable to the analysis of a limited set of biological samples. To close this gap between the traditional and the high-throughput procedures we have developed a method in which a condition-specific, genome-wide chromatin fragment library is produced and then used for locus-specific DNA fragment analysis. To validate the method, we used, as a test locus, the well-studied promoter of the divergently transcribed niiA and niaD genes coding for nitrate assimilation enzymes in Aspergillus. Additionally, we have used the condition-specific libraries to study nucleosomal positioning at two different loci, the promoters of the general nitrogen regulator areA and the regulator of secondary metabolism, aflR

N-Glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans

Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. β1,2-xylose and core α1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human β1,4-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIII), α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnTIV) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics

Community profiling and gene expression of fungal assimilatory nitrate reductases in agricultural soil

Although fungi contribute significantly to the microbial biomass in terrestrial ecosystems, little is known about their contribution to biogeochemical nitrogen cycles. Agricultural soils usually contain comparably high amounts of inorganic nitrogen, mainly in the form of nitrate. Many studies focused on bacterial and archaeal turnover of nitrate by nitrification, denitrification and assimilation, whereas the fungal role remained largely neglected. To enable research on the fungal contribution to the biogeochemical nitrogen cycle tools for monitoring the presence and expression of fungal assimilatory nitrate reductase genes were developed. To the ∼100 currently available fungal full-length gene sequences, another 109 partial sequences were added by amplification from individual culture isolates, representing all major orders occurring in agricultural soils. The extended database led to the discovery of new horizontal gene transfer events within the fungal kingdom. The newly developed PCR primers were used to study gene pools and gene expression of fungal nitrate reductases in agricultural soils. The availability of the extended database allowed affiliation of many sequences to known species, genera or families. Energy supply by a carbon source seems to be the major regulator of nitrate reductase gene expression for fungi in agricultural soils, which is in good agreement with the high energy demand of complete reduction of nitrate to ammonium