Dynamics of Bacterial Root Endophytes of Malus domestica Plants Grown in Field Soils Affected by Apple Replant Disease

Apple replant disease (ARD) is a worldwide problem for tree nurseries and orchards leading to reduced plant growth and fruit quality. The etiology of this complex phenomenon is poorly understood, but shifts of the bulk soil and rhizosphere microbiome seem to play an important role. Since roots are colonized by microbes from the rhizosphere, studies of the endophytic microbiome in relation to ARD are meaningful. In this study, culture-independent and culture-dependent approaches were used in order to unravel the endophytic root microbiome of apple plants 3, 7, and 12 months after planting in ARD-affected soil and ARD-unaffected control soil at two different field sites. Next to a high diversity of Pseudomonas in roots from all soils, molecular barcoding approaches revealed an increase in relative abundance of endophytic Actinobacteria over time in plants grown in ARD and control plots. Furthermore, several amplicon sequence variants (ASVs) linked to Streptomyces, which had been shown in a previous greenhouse ARD biotest to be negatively correlated to shoot length and fresh mass, were also detected in roots from both field sites. Especially in roots of apple plants from control soil, these Streptomyces ASVs increased in their relative abundance over time. The isolation of 150 bacterial strains in the culture-dependent approach revealed a high diversity of members of the genus Pseudomonas, confirming the data of the molecular barcoding approach. However, only partial overlaps were found between the two approaches, underlining the importance of combining these methods in order to better understand this complex disease and develop possible countermeasures. Overall, this study suggests a key role of Streptomyces in the etiology of ARD in the field. Copyright © 2022 Mahnkopp-Dirks, Radl, Kublik, Gschwendtner, Schloter and Winkelmann

Pseudomonas campi sp. nov., a nitrate-reducing bacterium isolated from grassland soil

A novel strain was isolated from grassland soil that has the potential to assimilate ammonium by the reduction of nitrate in the presence of oxygen. Whole genome sequence analysis revealed the presence of an assimilatory cytoplasmic nitrate reductase gene nasA and the assimilatory nitrite reductase genes nirBD which are involved in the sequential reduction of nitrate to nitrite and further to ammonium, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate represents a member of the genus Pseudomonas. The closest phylogenetic neighbours based on 16S rRNA gene sequence analysis are the type strains of Pseudomonas peli (98.17%) and Pseudomonas guineae (98.03%). In contrast, phylogenomic analysis revealed a close relationship to Pseudomonas alcaligenes. Computation of the average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) with the closest phylogenetic neighbours of S1-A32-2T revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. On the basis of these results, it was concluded that the soil isolate represents a novel species of the genus Pseudomonas, for which the name Pseu-domonas campi sp. nov. (type strain S1-A32-2T=LMG 31521T=DSM 110222T) is proposed

Early life determinants induce sustainable changes in the gut microbiome of six-year-old children

While the association between early life determinants and the development of the gut microbiome composition in infancy has been widely investigated, a potential persistent influence of early life determinants on the gut microbial community after its stabilization at later childhood remains largely unknown. Therefore, we aimed to identify the association between several early life determinants and the gut microbiome composition in six-year-old children from the LISA birth cohort. A total number of 166 fecal samples were analyzed using 16S rRNA gene-based barcoding to assess bacterial diversity pattern. The bacterial profiles were investigated for their association with maternal smoking during pregnancy, mode of delivery, breastfeeding, antibiotic treatment between one and two years of age, gender and socioeconomic status (SES). While alpha and beta diversity of the infants' gut microbiome remained unaffected, amplicon sequence variants (ASVs) annotated to Firmicutes and Actinobacteria responded to early life determinants, mostly to feeding practice and antibiotics use. ASVs associated to Bacteriodetes remained unaffected. Our findings indicate that early life determinants could have a long-term sustainable effect on the gut microflora of six-year-old children, however, associations with early life determinates are weaker than reported for infants

N2 gas flushing alleviates the loss of bacterial diversity and inhibits psychrotrophic Pseudomonas during the cold storage of bovine raw milk

Peer reviewe

Effects of industrial effluents containing moderate levels of antibiotic mixtures on the abundance of antibiotic resistance genes and bacterial community composition in exposed creek sediments

Environmental discharges of very high (mg/L) antibiotic levels from pharmaceutical production contributed to the selection, spread and persistence of antibiotic resistance. However, the effects of less antibiotic-polluted effluents (μg/L) from drug-formulation on exposed aquatic microbial communities are still scarce. Here we analyzed formulation effluents and sediments from the receiving creek collected at the discharge site (DW0), upstream (UP) and 3000 m downstream of discharge (DW3000) during winter and summer season. Chemical analyses indicated the largest amounts of trimethoprim (up to 5.08 mg/kg) and azithromycin (up to 0.39 mg/kg) at DW0, but sulfonamides accumulated at DW3000 (total up to 1.17 mg/kg). Quantitative PCR revealed significantly increased relative abundance of various antibiotic resistance genes (ARGs) against β-lactams, macrolides, sulfonamides, trimethoprim and tetracyclines in sediments from DW0, despite relatively high background levels of some ARGs already at UP site. However, only sulfonamide (sul2) and macrolide ARG subtypes (mphG and msrE) were still elevated at DW3000 compared to UP. Sequencing of 16S rRNA genes revealed pronounced changes in the sediment bacterial community composition from both DW sites compared to UP site, regardless of the season. Numerous taxa with increased relative abundance at DW0 decreased to background levels at DW3000, suggesting die-off or lack of transport of effluent-originating bacteria. In contrast, various taxa that were more abundant in sediments than in effluents increased in relative abundance at DW3000 but not at DW0, possibly due to selection imposed by high sulfonamide levels. Network analysis revealed strong correlation between some clinically relevant ARGs (e.g. blaGES, blaOXA, ermB, tet39, sul2) and taxa with elevated abundance at DW sites, and known to harbour opportunistic pathogens, such as Acinetobacter, Arcobacter, Aeromonas and Shewanella. Our results demonstrate the necessity for improved management of pharmaceutical and rural waste disposal for mitigating the increasing problems with antibiotic resistance

Simplification of soil biota communities impairs nutrient recycling and enhances above- and belowground nitrogen losses

Agriculture is a major source of nutrient pollution, posing a threat to the earth system functioning. Factors determining the nutrient use efficiency of plant-soil systems need to be identified to develop strategies to reduce nutrient losses while ensuring crop productivity. The potential of soil biota to tighten nutrient cycles by improving plant nutrition and reducing soil nutrient losses is still poorly understood. We manipulated soil biota communities in outdoor lysimeters, planted maize, continuously collected leachates, and measured N$_{2}$ O- and N$_{2}$ -gas emissions after a fertilization pulse to test whether differences in soil biota communities affected nutrient recycling and N losses. Lysimeters with strongly simplified soil biota communities showed reduced crop N (-20%) and P (-58%) uptake, strongly increased N leaching losses (+65%), and gaseous emissions (+97%) of N$_{2}$ O and N$_{2}$ . Soil metagenomic analyses revealed differences in the abundance of genes responsible for nutrient uptake, nitrate reduction, and denitrification that helped explain the observed nutrient losses. Soil biota are major drivers of nutrient cycling and reductions in the diversity or abundance of certain groups (e.g. through land-use intensification) can disrupt nutrient cycling, reduce agricultural productivity and nutrient use efficiency, and exacerbate environmental pollution and global warming

Pollution from azithromycin-manufacturing promotes macrolide-resistance gene propagation and induces spatial and seasonal bacterial community shifts in receiving river sediments

Effluents from antibiotic manufacturing may contain high concentrations of antibiotics, which are the main driving force behind the selection and spread of antibiotic resistance genes in the environment. However, our knowledge about the impact of such effluent discharges on the antibiotic resistome and bacterial communities is still limited. To gain insight into this impact, we collected effluents from an azithromycin-manufacturing industry discharge site as well as upstream and downstream sediments from the receiving Sava river during both winter and summer season. Chemical analyses of sediment and effluent samples indicated that the effluent discharge significantly increased the amount of macrolide antibiotics, heavy metals and nutrients in the receiving river sediments. Quantitative PCR revealed a significant increase of relative abundances of macrolideresistance genes and class 1 integrons in effluent- impacted sediments. Amplicon sequencing of 16S rRNA genes showed spatial and seasonal bacterial community shifts in the receiving sediments. Redundancy analysis and Mantel test indicated that macrolides and copper together with nutrients significantly correlated with community shift close to the effluent discharge site. The number of taxa that were significantly increased in relative abundance at the discharge site decreased rapidly at the downstream sites, showing the resilience of the indigenous sediment bacterial community. Seasonal changes in the chemical properties of the sediment along with changes in effluent community composition could be responsible for sediment community shifts between winter and summer. Altogether, this study showed that the discharge of pharmaceutical effluents altered physicochemical characteristics and bacterial community of receiving river sediments, which contributed to the enrichment of macrolide-resistance genes and integrons

Biological soil crusts on agricultural soils of mesic regions promote microbial cross-kingdom co-occurrences and nutrient retention

IntroductionBiological soil crusts (biocrusts) are known as biological hotspots on undisturbed, nutrient-poor bare soil surfaces and until now, are mostly observed in (semi-) arid regions but are currently poorly understood in agricultural systems. This is a crucial knowledge gap because managed sites of mesic regions can quickly cover large areas. Thus, we addressed the questions (i) if biocrusts from agricultural sites of mesic regions also increase nutrients and microbial biomass as their (semi-) arid counterparts, and (ii) how microbial community assemblage in those biocrusts is influenced by disturbances like different fertilization and tillage regimes.MethodsWe compared phototrophic biomass, nutrient concentrations as well as the abundance, diversity and co-occurrence of Archaea, Bacteria, and Fungi in biocrusts and bare soils at a site with low agricultural soil quality.Results and DiscussionBiocrusts built up significant quantities of phototrophic and microbial biomass and stored more nutrients compared to bare soils independent of the fertilizer applied and the tillage management. Surprisingly, particularly low abundant Actinobacteria were highly connected in the networks of biocrusts. In contrast, Cyanobacteria were rarely connected, which indicates reduced importance within the microbial community of the biocrusts. However, in bare soil networks, Cyanobacteria were the most connected bacterial group and, hence, might play a role in early biocrust formation due to their ability to, e.g., fix nitrogen and thus induce hotspot-like properties. The microbial community composition differed and network complexity was reduced by conventional tillage. Mineral and organic fertilizers led to networks that are more complex with a higher percentage of positive correlations favoring microbe-microbe interactions. Our study demonstrates that biocrusts represent a microbial hotspot on soil surfaces under agricultural use, which may have important implications for sustainable management of such soils in the future

Molecular Barcoding Reveals the Genus Streptomyces as Associated Root Endophytes of Apple (Malus domestica) Plants Grown in Soils Affected by Apple Replant Disease

Apple replant disease (ARD) occurs when apple is repeatedly planted at the same site, leading to growth reductions and losses in fruit yield and quality. Up to now, the etiology has been poorly understood; however, soil (micro)biota are known to be involved. Because endophytes often colonize plants via the rhizosphere, this study aimed at comparing the bacterial endophytic root microbiome in plants growing in ARD-affected and unaffected soils from three different sites based on greenhouse biotests using a molecular barcoding approach. The initial endophytic microbiome of the starting material (in vitro propagated plants of the apple rootstock M26) did not significantly affect the overall richness and diversity of the endophytic community in plants after 8 weeks of growth in the respective soils but some genera of the initial microbiome managed to establish in apple roots. Proteobacteria was the dominant phylum in all samples. No differences in diversity or number of amplicon sequence variants (ASVs) between plants grown in ARD soil and unaffected soil was observed. However, several ASVs of high abundance uniquely found in plants grown in ARD-affected soils were Streptomyces spp. In soil from all three sites, these Streptomyces spp. were negatively correlated with plant growth parameters. Future inoculation experiments using selected Streptomyces isolates have to prove whether bacteria from this genus are opportunists or part of the ARD complex. For the first time, the bacterial endophytic community of apple roots grown in ARD-affected soils was characterized, which will help us to understand the etiology of ARD and develop countermeasures

Long-term ferrocyanide application via deicing salts promotes the establishment of Actinomycetales assimilating ferrocyanide-derived carbon in soil

Cyanides are highly toxic and produced by various microorganisms as defence strategy or to increase their competitiveness. As degradation is the most efficient way of detoxification, some microbes developed the capability to use cyanides as carbon and nitrogen source. However, it is not clear if this potential also helps to lower cyanide concentrations in roadside soils where deicing salt application leads to significant inputs of ferrocyanide. The question remains if biodegradation in soils can occur without previous photolysis. By conducting a microcosm experiment using soils with/without pre-exposition to road salts spiked with C-13-labelled ferrocyanide, we were able to confirm biodegradation and in parallel to identify bacteria using ferrocyanide as C source via DNA stable isotope probing (DNA-SIP), TRFLP fingerprinting and pyrosequencing. Bacteria assimilating C-13 were highly similar in the pre-exposed soils, belonging mostly to Actinomycetales (Kineosporia, Mycobacterium, Micromonosporaceae). In the soil without pre-exposition, bacteria belonging to Acidobacteria (Gp3, Gp4, Gp6), Gemmatimonadetes (Gemmatimonas) and Gammaproteobacteria (Thermomonas, Xanthomonadaceae) used ferrocyanide as C source but not the present Actinomycetales. This indicated that (i) various bacteria are able to assimilate ferrocyanide-derived C and (ii) long-term exposition to ferrocyanide applied with deicing salts leads to Actinomycetales outcompeting other microorganisms for the use of ferrocyanide as C source