Microsatellite-Based Quantification Method to Estimate Biomass of Endophytic Phialocephala Species in Strain Mixtures

Fungi of the Phialocephala fortinii sensu lato-Acephala applanata species complex (PAC) are ubiquitous endophytic colonizers of tree roots in which they form genotypically diverse communities. Measurement of the colonization density of each of the fungal colonizers is a prerequisite to study the ecology of these communities. Up to now, there is no method readily available for the quantification of PAC strains co-colonizing the same root. The new DNA quantification method presented here is based on the amplification of microsatellites by competitive polymerase chain reaction (PCR). The method proved to be suitable to detect and quantify at least two strains within one single sample by the addition of a known amount of mycelium of a reference strain before DNA extraction. The method exploits the correlation between the reference/target ratio of light emitted during microsatellite detection (peak ratio) and the reference/target ratio of mycelial weights to determine the biomass of the target strain. Hence, calibration curves were obtained by linear regression of the peak ratios on the weight ratios for different mixtures of reference and target strains. The slopes of the calibration curves and the coefficients of determination were close to 1, indicating that peak ratios are good predictors of weight ratios. Estimates of fungal biomass in mycelial test mixtures of known composition laid within the 95% prediction interval and deviated on average by 16% (maximally 50%) from the true biomass. On average, 3-6% of the root biomass of Norway spruce seedlings consisted of mycelial biomass of either one of two inoculated PAC strains. Biomass estimates obtained by real-time quantitative PCR were correlated with the estimates obtained by the microsatellite-based method, but variation between the two estimates from the same root was high in some samples. The microsatellite-based DNA quantification method described here is currently the best method for strainwise estimation of endophytic biomass of PAC fungi in small root sample

Control of pathogenic PAC strains by non-pathogenic PAC strains in planta does not correlate with higher competitiveness of non-pathogenic PAC strains ex planta

Ascomycetes of the Phialocephala fortinii s.l.—Acephala applanata species complex (PAC) are frequent root endophytes of forest trees. Roots are colonized by multiple PAC genotypes that interact, and recent findings indicate that adverse effects on plant performance caused by pathogenic PAC strains are attenuated by non-pathogenic PAC strains. However, it was not known if this "self-control” works only in planta, or also ex planta, i.e., prior to infection during saprotrophic life of the PAC. Interactions between PAC strains were therefore studied in a plant-free system on malt extract agar. The mycelia of two pathogenic (A and T1) and two non-pathogenic (B and C) PAC strains were mixed pairwise 5:1, 1:1 and 1:5 (fresh weight ratios) and incubated at 15 and 25°C. Mycelial biomass of each strain was measured after 2 and 8 weeks. The combination of strains and the mixture ratio had a significant effect on strain biomass, whereas temperature influenced only the biomass of pathogenic strain T1. Biomass production of strain T1 was inhibited by all other strains, whereas biomass production of the other pathogenic strain A was significantly stimulated by the two non-pathogenic strains. This contrasts strongly with results from a previous experiment in planta using strains A, B and C, because the two non-pathogenic PAC strains successfully inhibited the pathogenic strain, probably by space occupation or the induction of host resistance. Therefore, it is impossible to predict the outcome of PAC-PAC interactions in planta based on the results gained from interactions ex planta

Pesticides in Agricultural Soils: Major Findings from Various Monitoring Campaigns in Switzerland

Synthetic pesticides are widely applied in modern agriculture, where they are used against diseases, pests, and weeds to secure crop yield and quality. However, their intensive application has led to widespread contamination of the environment, including soils. Due to their inherent toxicity, they might pose a risk to soil health by causing harm to non-target organisms and disrupting ecosystem services in both agricultural and other exposed soils. Following the Swiss National Action Plan on the reduction of pesticide risks, Agroscope has conducted several soil monitoring studies that are briefly presented here. All of them resort to different multi-residue trace analytical approaches to simultaneously quantify up to about 150 modern pesticides by either accelerated solvent, or Quick, Easy, Cheap, Efficient, Rugged, Safe (QuEChERS) extraction, followed by separation and detection with liquid chromatography-triple quadrupole mass spectrometry. While partly still in progress, our investigations led to the following major findings this far: Multiple pesticides are commonly present in soils, with individual concentrations in agricultural soils often reaching up to a few tens of µg/kg. Pesticide occurrence and concentrations in agricultural soils primarily depend on land use, land use history and cultivated crops. Pesticides can prevail much longer than predicted by their half-lives, and were found in soils even decades after conversion from conventional to organic farming. Corresponding residual fractions can be in the order of a few percent of the originally applied amounts. We further found negative associations of pesticide residues with the abundance of beneficial soil life, underpinning their potential risk to the fertility of agricultural soils. Traces of pesticides are also detected in soils to which they were never applied, indicating contamination, e.g., via spray drift or atmospheric deposition. These results confirm the general notion of both scientists and legislators that prospective risk assessments (RA; as executed during registration and use authorization) should be confirmed and adjusted by retrospective RA (e.g., by environmental monitoring studies of currently used compounds) to jointly lead to an overall reduced environmental risk of pesticides

Pesticides in Agricultural Soils: Major Findings from Various Monitoring Campaigns in Switzerland

Synthetic pesticides are widely applied in modern agriculture, where they are used against diseases, pests, and weeds to secure crop yield and quality. However, their intensive application has led to widespread contamination of the environment, including soils. Due to their inherent toxicity, they might pose a risk to soil health by causing harm to non-target organisms and disrupting ecosystem services in both agricultural and other exposed soils. Following the Swiss National Action Plan on the reduction of pesticide risks, Agroscope has conducted several soil monitoring studies that are briefly presented here. All of them resort to different multi-residue trace analytical approaches to simultaneously quantify up to about 150 modern pesticides by either accelerated solvent, or Quick, Easy, Cheap, Efficient, Rugged, Safe (QuEChERS) extraction, followed by separation and detection with liquid chromatography-triple quadrupole mass spectrometry. While partly still in progress, our investigations led to the following major findings this far: Multiple pesticides are commonly present in soils, with individual concentrations in agricultural soils often reaching up to a few tens of µg/kg. Pesticide occurrence and concentrations in agricultural soils primarily depend on land use, land use history and cultivated crops. Pesticides can prevail much longer than predicted by their half-lives, and were found in soils even decades after conversion from conventional to organic farming. Corresponding residual fractions can be in the order of a few percent of the originally applied amounts. We further found negative associations of pesticide residues with the abundance of beneficial soil life, underpinning their potential risk to the fertility of agricultural soils. Traces of pesticides are also detected in soils to which they were never applied, indicating contamination, e.g., via spray drift or atmospheric deposition. These results confirm the general notion of both scientists and legislators that prospective risk assessments (RA; as executed during registration and use authorization) should be confirmed and adjusted by retrospective RA (e.g., by environmental monitoring studies of currently used compounds) to jointly lead to an overall reduced environmental risk of pesticides

Pesticides in Agricultural Soils: Major Findings from Various Monitoring Campaigns in Switzerland

Synthetic pesticides are widely applied in modern agriculture, where they are used against diseases, pests, and weeds to secure crop yield and quality. However, their intensive application has led to widespread contamination of the environment, including soils. Due to their inherent toxicity, they might pose a risk to soil health by causing harm to non-target organisms and disrupting ecosystem services in both agricultural and other exposed soils. Following the Swiss National Action Plan on the reduction of pesticide risks, Agroscope has conducted several soil monitoring studies that are briefly presented here. All of them resort to different multi-residue trace analytical approaches to simultaneously quantify up to about 150 modern pesticides by either accelerated solvent, or Quick, Easy, Cheap, Efficient, Rugged, Safe (QuEChERS) extraction, followed by separation and detection with liquid chromatography-triple quadrupole mass spectrometry. While partly still in progress, our investigations led to the following major findings this far: Multiple pesticides are commonly present in soils, with individual concentrations in agricultural soils often reaching up to a few tens of µg/kg. Pesticide occurrence and concentrations in agricultural soils primarily depend on land use, land use history and cultivated crops. Pesticides can prevail much longer than predicted by their half-lives, and were found in soils even decades after conversion from conventional to organic farming. Corresponding residual fractions can be in the order of a few percent of the originally applied amounts. We further found negative associations of pesticide residues with the abundance of beneficial soil life, underpinning their potential risk to the fertility of agricultural soils. Traces of pesticides are also detected in soils to which they were never applied, indicating contamination, e.g., via spray drift or atmospheric deposition. These results confirm the general notion of both scientists and legislators that prospective risk assessments (RA; as executed during registration and use authorization) should be confirmed and adjusted by retrospective RA (e.g., by environmental monitoring studies of currently used compounds) to jointly lead to an overall reduced environmental risk of pesticides

Analysis of the <i>Phialocephala subalpina</i> Transcriptome during Colonization of Its Host Plant <i>Picea abies</i>

<div><p>Background</p><p><i>Phialocephala subalpina</i> belongs to the <i>Phialocephala fortinii</i> s.l.–<i>Acepphala applanata</i> species complex (PAC) forming one of the major groups belonging to the dark septate endophytes (DSE). Depending on the strain, PAC was shown to form neutral to pathogenic associations with its host plant <i>Picea abies</i>. To understand PACs lifestyle we investigated the effect of presence/absence of <i>Picea abies</i> on the transcriptome of strain 6_70_1.</p><p>Materials and Methods</p><p>PAC strain 6_70_1 was grown in liquid Pachlewski media either induced by its host plant <i>Picea abies</i> or without host plant as a control. Mycelia were harvested in a time course (1, 2, 3, 4, 7, 11, 18 days) with and without induction by the host plant and the fungal transcriptome revealed by Illumina sequencing. Differential gene expression analysis over the time course comparing control and treatment at each time point using the ‘edgeR glm approach’ and a gene enrichment analysis using GO categories were performed.</p><p>Results</p><p>The three main functional groups within differentially expressed genes were ‘metabolism’, ‘transport’ and ‘cell rescue, defense and virulence’. Additionally, genes especially involved in iron metabolism could be detected by gene set enrichment analysis.</p><p>Conclusion</p><p>In conclusion, we found PAC strain 6_70_1 to be metabolically very active during colonization of its host plant <i>Picea abies</i>. A major shift in functional groups over the time course of this experiment could not be observed but GO categories which were found to be enriched showed different emphasis depending in the day post induction.</p></div

Control of pathogenic PAC strains by non-pathogenic PAC strains in planta does not correlate with higher competitiveness of non-pathogenic PAC strains ex planta

Ascomycetes of the Phialocephala fortinii s.l.—Acephala applanata species complex (PAC) are frequent root endophytes of forest trees. Roots are colonized by multiple PAC genotypes that interact, and recent findings indicate that adverse effects on plant performance caused by pathogenic PAC strains are attenuated by non-pathogenic PAC strains. However, it was not known if this “self-control” works only in planta, or also ex planta, i.e., prior to infection during saprotrophic life of the PAC. Interactions between PAC strains were therefore studied in a plant-free system on malt extract agar. The mycelia of two pathogenic (A and T1) and two non-pathogenic (B and C) PAC strains were mixed pairwise 5:1, 1:1 and 1:5 (fresh weight ratios) and incubated at 15 and 25 °C. Mycelial biomass of each strain was measured after 2 and 8 weeks. The combination of strains and the mixture ratio had a significant effect on strain biomass, whereas temperature influenced only the biomass of pathogenic strain T1. Biomass production of strain T1 was inhibited by all other strains, whereas biomass production of the other pathogenic strain A was significantly stimulated by the two non-pathogenic strains. This contrasts strongly with results from a previous experiment in planta using strains A, B and C, because the two non-pathogenic PAC strains successfully inhibited the pathogenic strain, probably by space occupation or the induction of host resistance. Therefore, it is impossible to predict the outcome of PAC-PAC interactions in planta based on the results gained from interactions ex planta.ISSN:1617-416XISSN:1861-895

Top 20 differentially expressed genes.

<p>Heat map depicting the Top 20 genes including their main functional categories. Gene expression in blue shows down-regulated and in orange up-regulated genes. Genes were clustered by their expression pattern. The transcription level is depicted in logFC values. Genes displayed in blue are downregulated, therefore expressed in favor of the control and genes depicted in orange are expressed in favor of the treatment. Count corresponds to the number of reads covering the gene model at each timepoint.</p

FunCat functional main categories in DE genes and their number summarized over all days.

<p>FunCat functional main categories in DE genes and their number summarized over all days.</p