Short-Term Impact of Recycling-Derived Fertilizers on Their P Supply for Perennial Ryegrass (Lolium perenne).

Various nutrient recycling technologies are currently under development in order to alleviate the dependency of non-renewable raw material for the production of mineral phosphorus fertilizers commonly used in agriculture. The resulting products, such as struvites and ashes, need to be assessed for their application as so-called recycling-derived fertilizers (RDFs) in the agricultural sector prior to commercialization. Here, we conducted a short-term (54 days) trial to investigate the impact of different phosphorus fertilizers on plant growth and the soil P cycling microbiota. Lolium perenne was grown with application of superphosphate (SP) as inorganic fertilizer, two ashes (poultry litter ash (PLA) and sewage sludge ash (SSA)), and two struvites (municipal wastewater struvite (MWS) and commercial CrystalGreen® (CGS)) applied at 20 and 60 kg P ha-1 in four replicates. A P-free control (SP0) was also included in the trial. Struvite application increased plant dry weights, and available P acid phosphatase activity was significantly improved for struvites at the high P application rate. The ash RDFs showed a liming effect at 60 kg P ha-1, and PLA60 negatively affected acid phosphatase activity, while PLA20 had significantly lower phoD copy numbers. P mobilization from phosphonates and phytates was not affected. TCP solubilization was negatively affected by mineral SP fertilizer application at both P concentrations. The bacterial (16S and phoD) communities were only marginally affected by the tested P fertilizers. Overall, struvites appeared to be a suitable substitute for superphosphate fertilization for Irish L. perenne pastures.

Arbuscular Mycorrhiza Support Plant Sulfur Supply through Organosulfur Mobilizing Bacteria in the Hypho- and Rhizosphere.

This study aimed to elucidate the role of bacteria colonising mycorrhizal hyphae in organically bound sulfur mobilisation, the dominant soil sulfur source that is not directly plant available. The effect of an intact mycorrhizal symbiosis with access to stable isotope organo-34S enriched soils encased in 35 µm mesh cores was tested in microcosms with Agrostis stolonifera and Plantago lanceolata. Hyphae and associated soil were sampled from static mesh cores with mycorrhizal ingrowth and rotating mesh cores that exclude mycorrhizal ingrowth as well as corresponding rhizosphere soil, while plant shoots were analysed for 34S uptake. Static cores increased uptake of 34S at early stages of plant growth when sulfur demand appeared to be high and harboured significantly larger populations of sulfonate mobilising bacteria. Bacterial and fungal communities were significantly different in the hyphospheres of static cores when compared to rotating cores, not associated with plant hosts. Shifts in bacterial and fungal communities occurred not only in rotated cores but also in the rhizosphere. Arylsulfatase activity was significantly higher in the rhizosphere when cores stayed static, while atsA and asfA gene diversity was distinct in the microcosms with static and rotating cores. This study demonstrated that AM symbioses can promote organo-S mobilization and plant uptake through interactions with hyphospheric bacteria, enabling AM fungal ingrowth into static cores creating a positive feedback-loop, detectable in the microbial rhizosphere communities.

Cultivation Conditions of Spinach and Rocket Influence Epiphytic Growth of Listeria monocytogenes.

Leafy vegetables are associated with Listeriosis outbreaks due to contamination with Listeria monocytogenes. To date, contradictory findings were reported on spinach, rocket, and kale, where some studies reported growth of L. monocytogenes, while others did not. Thus, the current study investigated the reason for conflicting findings by producing leafy vegetables, where cultivation factors were known for growth potential studies. Of all polytunnel produce, kale Nero di Toscana demonstrated the highest growth potential (2.56 log cfu g−1), followed by spinach F1 Cello (1.84 log cfu g−1), rocket Buzz (1.41 log cfu g−1), spinach F1 Trumpet (1.37 log cfu g−1), and finally rocket Esmee (1.23 log cfu g−1). Thus, plant species and variety influenced L. monocytogenes growth potentials. Moreover, significantly lower growth potentials of 0.3 log cfu g−1 were identified when rocket Buzz was cultivated in open fields (1.11 log cfu g−1) instead of a polytunnel. The opposite effect was observed for spinach F1 Trumpet, where growth potentials increased significantly by 0.84 log cfu g−1 when cultivated in open fields (2.21 log cfu g−1). Furthermore, a significant seasonality effect between batches was found (p < 0.05). This study revealed that spinach and rocket cultivation conditions are at least co-factors in the reporting of differing growth potentials of L. monocytogenes across literature and should be considered when conducting future growth potential studies.

International Spread of Multidrug-Resistant Rhodococcus equi.

A multidrug-resistant clone of the animal and human pathogen Rhodococcus equi, MDR-RE 2287, has been circulating among equine farms in the United States since the 2000s. We report the detection of MDR-RE 2287 outside the United States. Our finding highlights the risk for MDR-RE spreading internationally with horse movements.

Design and Construction of Repacked Soil Columns for Measuring Solute Transport, Plant Growth and Soil Biota.

Researchers face a number of challenges in the construction of soil columns which can affect the outcome of their experiments. The use of intact soil cores closely mimics actual field conditions. However, the excavation of large intact soil cores is a time-consuming, labor-intensive process and may lead to soil compaction that would influence the solute transport behavior of the soil column. Repacked soil columns are used as an option to circumvent these challenges of intact soil cores. However, repacked soil columns also have their limitations and introduce other challenges. Here, we present a step by step procedure for the design of repacked soil columns to achieve a realistic bulk density, prevent preferential flow paths, and ensure hydraulic connectivity between soil layers. This protocol will be beneficial to Soil Scientists, Hydrologists and other Environmental Scientists utilizing repacked soil columns.

Growth Potential of Listeria monocytogenes on Refrigerated Spinach and Rocket Leaves in Modified Atmosphere Packaging.

Minimally processed ready-to-eat (RTE) vegetables are increasingly consumed for their health benefits. However, they also pose a risk of being ingested with food-borne pathogens. The present study investigated the ability of RTE spinach and rocket to support the growth of Listeria monocytogenes as previous studies provided contradicting evidence. Findings were compared to growth on iceberg lettuce that has repeatedly been shown to support growth. Products were inoculated with a three-strain mix of L. monocytogenes at 10 and 100 cfu g-1 and stored in modified atmosphere (4 kPa O2, 8 kPa CO2) at 8 °C over 7-9 days. Spinach demonstrated the highest growth potential rate of 2 to 3 log10 cfu g-1 over a 9-day period with only marginal deterioration in its visual appearance. Growth potential on rocket was around 2 log10 cfu g-1 over 9 days with considerable deterioration in visual appearance. Growth potential of iceberg lettuce was similar to that of rocket over a 7-day period. Growth curves fitted closely to a linear growth model, indicating none to limited restrictions of growth over the duration of storage. The high growth potentials of L. monocytogenes on spinach alongside the limited visual deterioration highlight the potential risks of consuming this raw RTE food product when contaminated.

Sulfatase Assay to Determine Influence of Plants on Microbial Activity in Soil.

Sulfatase activity is often used as a measure of the activity of soil microorganisms. It is thus a suitable tool to investigate the response of microbes to plants. Here we present a method to determine the influence of various Arabidopsis genotypes on the function of soil microbiota using the sulfatase as a quantitative measure. We grew the plants in soil/sand mix under control conditions and measured the sulfatase activity in soil using a spectrophotometric determination of the product. This protocol can be used to test the contribution of individual genes to control of microbiome assembly through analysis of mutants as well as the influence of environment on plant-microbe interactions.

Sulfate fertilization supports growth of ryegrass in soil columns but changes microbial community structures and reduces abundances of nematodes and arbuscular mycorrhiza.

The increased use of sulfate fertilizers to compensate for soil sulphur (S) limitation in agricultural soils may affect soil microbes and micro-fauna involved in S mobilization. Here, columns with podzolic soil material and ryegrass (Lolium perenne) were fertilized with 0, 5, 10 and 20 kg ha-1 (S0/S5/S10/S20) inorganic sulfate-S alongside a full complement of other nutrients. In the S10 and S20 columns, significantly higher amounts of sulfate were present in soil solution. After two grass cuts (14 weeks in total), there was a significant decrease in arylsulfatase activity, bacterial-feeding nematode abundances and mycorrhizal colonization in the S10 and S20 columns compared to the S0. Bacterial, fungal and AM community structures shifted significantly across the treatments. After final harvest, the S10 and S20 columns had significantly higher grass dry matter yield and uptake of S, N, K, Ca and Mg compared to the S0. While the overall bacterial diversity was reduced in the S20 treatment, abundance (asfA) and diversity (ssuD and atsA) of bacterial genes involved in S cycling were not significantly affected by one-time sulfate fertilization. These results indicate that short-term sulfate fertilization benefits to plant growth outweighed the negative feedback from parts of the soil biota. To improve nutrient use efficiencies in a sustainable manner, future studies should consider alternative S fertilizers which may be beneficial to both, the soil biota and plants in the long-term.

One-time phosphate fertilizer application to grassland columns modifies the soil microbiota and limits its role in ecosystem services.

This study evaluated the effect of one-time phosphate fertilization on the soil microbiota, its cycling of phosphorus (P) and grass growth. Soil columns were established in a greenhouse using a P-limited Irish soil (index 1), planted with Lolium perenne and fertilized with 0 (control), 5 (quarter), 10 (half) and 20 (full)kgPha-1 as inorganic phosphate. Only traces of phosphate in soil solution were detected over the 14week experiment, even after phosphate fertilization. Grass dry matter yield between treatments was not significantly different. Full phosphate fertilization significantly reduced the arbuscular mycorrhization (AM) rate, bacterial- and fungal-feeding nematode population, bacterial phoD gene abundance, but increased alkaline and acid phosphatase activities at the time of harvest. Full and half P treatments significantly shifted the bacterial, fungal and AM community structures compared to the control. Furthermore, the control had a significantly higher relative abundance of bacterial genera including Bacillus, Bradyrhizobium, Paenibacillus, Nocardioides and Balneimonas, that have been associated with P mobilization in the past, when compared to the full phosphate treatment. These results suggest that a positive effect of a single phosphate application on plant growth in a soil can be cancelled out by its negative effect on the soil microbiota and their ecosystem services.

Inoculation density is affecting growth conditions of Listeria monocytogenes on fresh cut lettuce.

Listeria monocytogenes is a particular risk for the ready-to-eat food sector because of its ability to grow in various environmental conditions. In the literature, growth and survival of L. monocytogenes on food is tested using inoculation densities ranging from less than 102 to over 105 CFU g-1. Inoculation densities on food have been rarely tested as a factor for growth. In this study, inoculation densities from 102 to 105 of L. monocytogenes were tested on iceberg lettuce (Lactuca sativa) in modified atmospheres and air in model packages at 4 and 8 °C to identify any potential inoculation density effects. On days 0, 2, 5 and 7, L. monocytogenes was extracted from the lettuce surface and enumerated via selective media. The resulting growth curves identified a significant inoculation density effect at 4 and 8 °C with significantly higher amounts of growth (1-2 logs) when lettuce was inoculated at 102 CFU g-1 as opposed to 104 and 105 CFU g-1. In contrast, the use of different atmospheres had limited influence on growth of L. monocytogenes. In conclusion, greater emphasis on inoculation density of L. monocytogenes should be taken in inoculation experiments when confirmation of growth or the efficacies of growth inhibiting treatments are tested on ready-to-eat food such as lettuce.

A 3-year multi-food study of the presence and persistence of Listeria monocytogenes in 54 small food businesses in Ireland.

The problem of assessing the occurrence of the food-borne pathogen Listeria monocytogenes in the food chain, and therefore the risk of exposure of the human population, is often challenging because of the limited scope of some studies. In this study the occurrence of L. monocytogenes in food from four major food groups, dairy products, meats, seafood and vegetables, and associated food processing environments in Ireland was studied over a three-year period. Fifty-four small food businesses participated in the study and sent both food and environmental samples every 2months between 2013 and 2015. L. monocytogenes was isolated using the ISO11290 standard method. Confirmation of L. monocytogenes and identification of serogroups were achieved using a multiplex PCR assay, and for some isolates serotype was determined using commercial antisera. Pulsed- field gel electrophoresis (PFGE) analysis was performed on all isolates allowing the relatedness of isolates from different food businesses to be compared nationwide. In total, 86 distinct pulsotypes were identified. The overall occurrence of L. monocytogenes in food samples was 4.2%, while in environmental samples it was 3.8%. In general, the occurrence of L. monocytogenes in food businesses decreased over the course of the study, presumably reflecting increased awareness and vigilance. The majority of the pulsotypes detected were unique to a particular food group (63/86), while only three pulsotypes were found in all four food groups investigated. The highest occurrence in food was found in the meat category (7.5%) while seafood had the lowest rate of occurrence (1.8%). Seventeen of the pulsotypes detected in the study were persistent, where persistence was defined as repeated isolation from a single facility with a minimum time interval of 6months. Using PFGE, 11 of the pulsotypes identified in this study were indistinguishable from those of 11 clinical isolates obtained from patients in Ireland over the last 4years, highlighting the fact that these pulsotypes are capable of causing disease. Overall, the study shows the diversity of L. monocytogenes strains in the Irish food chain and highlights the ability of many of these strains to persist in food processing environments. The finding that a significant proportion of these pulsotypes are also found in clinical settings highlights the need for continued vigilance by food producers, including frequent sampling and typing of isolates detected.

The role of sulfur- and phosphorus-mobilizing bacteria in biochar-induced growth promotion of Lolium perenne.

Plants rely on microorganisms to mobilize organically and inorganically bound sulfur (S) and phosphorus (P) in which the plant can then readily utilize. The aim of this study was to investigate the role of S- and P-mobilizing bacteria in plant growth promotion in biochar-amended soil, which has been rarely investigated so far. Pot experiments of Lolium perenne were established on S and P limited soil with 1% or 2% biochar (Miscanthus × giganteus) or without biochar (control) for a period of 126 days. Both biochar amendments resulted in significant plant growth promotion. Rhizobacteria capable of growing with (1) S from aromatic sulfonates, (2) P from phosphate esters, (3) P from phosphonates, and (4) P from tri-calcium phosphates as sole source of S or P, respectively, were significantly more abundant in the biochar treatments. 16S rRNA gene-based rhizobacteria community analysis revealed a significant biochar treatment effect. Abundance of nematodes feeding on bacteria was also significantly increased in the biochar treatments. Diversity analysis of rhizospheric asfA and phnJ genes revealed broad sequence diversities in bacterial sulfonate and phosphonate-mineralizing capabilities. These findings suggest that biochar amendment enhances microbially mediated nutrient mobilization of S and P resulting in improved plant growth.

Profiling the diversity of microbial communities with single-strand conformation polymorphism (SSCP).

Genetic fingerprinting techniques for microbial community analysis have evolved over the last decade into standard applications for efficient and fast differentiation of microbial communities based on their diversity. These techniques commonly analyze the diversity of PCR products amplified from extracted environmental DNA usually utilizing primers hybridizing to suspected conserved regions of the targeted genes. In comparison to the more commonly applied terminal restriction fragment length polymorphism (TRFLP) or denaturing gradient gel electrophoresis (DGGE) techniques, the here-described single-strand conformation polymorphism (SSCP) fingerprinting technique features some advantageous key characteristics. (1) Primers for the polymerase chain reaction (PCR) do only need minimal 5'-end alterations; (2) SSCP is adaptable to high throughput applications in automated sequencers; and (3) a second dimension in the SSCP gel electrophoresis can be implemented to obtain high resolution 2D gels. One central key requirement for SSCP gel electrophoresis is a tight temperature control. Gels that run at different temperatures will produce entirely different fingerprints. This can be exploited for an improved analysis of highly diverse communities by running the same template at different temperatures or by 2D-SSCP gel electrophoresis.

The role of bacteria and mycorrhiza in plant sulfur supply.

Plant growth is highly dependent on bacteria, saprophytic, and mycorrhizal fungi which facilitate the cycling and mobilization of nutrients. Over 95% of the sulfur (S) in soil is present in an organic form. Sulfate-esters and sulfonates, the major forms of organo-S in soils, arise through deposition of biological material and are transformed through subsequent humification. Fungi and bacteria release S from sulfate-esters using sulfatases, however, release of S from sulfonates is catalyzed by a bacterial multi-component mono-oxygenase system. The asfA gene is used as a key marker in this desulfonation process to study sulfonatase activity in soil bacteria identified as Variovorax, Polaromonas, Acidovorax, and Rhodococcus. The rhizosphere is regarded as a hot spot for microbial activity and recent studies indicate that this is also the case for the mycorrhizosphere where bacteria may attach to the fungal hyphae capable of mobilizing organo-S. While current evidence is not showing sulfatase and sulfonatase activity in arbuscular mycorrhiza, their effect on the expression of plant host sulfate transporters is documented. A revision of the role of bacteria, fungi and the interactions between soil bacteria and mycorrhiza in plant S supply was conducted.

Degradation of benzo[a]pyrene by bacterial isolates from human skin.

Polycyclic aromatic hydrocarbons (PAHs) are some of the most widespread xenobiotic pollutants, with the potentially carcinogenic high-molecular-weight representatives being of particular interest. However, while in eukaryotes, the cytochrome P450 (CYP)-mediated activation of benzo[a]pyrene (B[a]P) has become a model for metabolism-mediated carcinogenesis, the oxidative degradation of B[a]P by microorganisms is less well studied. This should be reason for concern as the human organ most exposed to environmental PAHs is the skin, which at the same time is habitat to a most diverse population of microbial commensals. Yet, nothing is known about the skin's microbiome potential to metabolise B[a]P. This study now reports on the isolation of 21 B[a]P-degrading microorganisms from human skin, 10 of which were characterised further. All isolates were able to degrade B[a]P as sole source of carbon and energy, and degradation was found to be complete in at least four isolates. Substrate metabolism involved two transcripts that encode a putative DszA/NtaA-like monooxygenase and a NifH-like reductase, respectively. Analysis of the 16S-rRNA genes showed that the B[a]P-degrading isolates comprise Gram(+) as well as Gram(-) skin commensals, with Micrococci being predominant. Moreover, microbial B[a]P-degradation was detected on all volunteers probed, indicating it to be a universal feature of the skin's microbiome.

Bacterial communities established in bauxite residues with different restoration histories.

Bauxite residue is the alkaline byproduct generated when alumina is extracted from bauxite ores and is commonly deposited in impoundments. These sites represent hostile environments with increased salinity and alkalinity and little prospect of revegetation when left untreated. This study reports the establishment of bacterial communities in bauxite residues with and without restoration amendments (compost and gypsum addition, revegetation) in samples taken in 2009 and 2011 from 0 to 10 cm depth. DNA fingerprint analysis of bacterial communities based on 16S rRNA gene fragments revealed a significant separation of the untreated site and the amended sites in both sampling years. 16S amplicon analysis (454 FLX pyrosequencing) revealed significantly lower alpha diversities in the unamended in comparison to the amended sites and hierarchical clustering separated the unamended site from the amended sites. The taxonomic analysis revealed that the restoration resulted in the accumulation of bacterial populations typical for soils including Acidobacteriaceae, Nitrosomonadaceae, and Caulobacteraceae. In contrast, the unamended site was dominated by taxonomic groups including Beijerinckiaceae, Xanthomonadaceae, Acetobacteraceae, and Chitinophagaceae, repeatedly associated with alkaline salt lakes and sediments. While bacterial communities developed in the initially sterile bauxite residue, only the restoration treatments created diverse soil-like bacterial communities alongside diverse vegetation on the surface.

Shifts in desulfonating bacterial communities along a soil chronosequence in the forefield of a receding glacier.

Forefields of receding glaciers are unique and sensitive environments representing natural soil chronosequences, where sulfate availability is assumed to be a limiting factor. Bacterial mineralization of organosulfur is an important sulfate-providing process in soils. We analyzed the diversity of sulfonate-desulfurizing (desulfonating) bacteria in the Damma glacier forefield on the basis of the key gene asfA by terminal restriction fragment length polymorphism and clone libraries. The community structure and sequence diversity of desulfonating bacteria differed significantly between forefield soils deglaciated in the 1990s and the 1950s. Soil age had a strong effect on the desulfonating rhizosphere communities of Agrostis rupestris, but only a slight impact on the ones from Leucanthemopsis alpina. AsfA affiliated to Polaromonas sp. was predominantly found in the more recent ice-free soils and the corresponding rhizospheres of A. rupestris, while a group of unidentified sequences was found to be dominating the matured soils and the corresponding rhizospheres of A. rupestris. The desulfonating bacterial diversity was not affected by varying levels of sulfate concentrations. The level of asfA diversity in recently deglaciated soils suggests that desulfonating bacteria are a critical factor in sulfur cycling, with defined groups dominating at different stages of soil formation.

Sulfonate desulfurization in Rhodococcus from wheat rhizosphere communities.

Organically bound sulfur makes up about 90% of the total sulfur in soils, with sulfonates often the dominant fraction. Actinobacteria affiliated to the genus Rhodococcus were able to desulfonate arylsulfonates in wheat rhizospheres from the Broadbalk long-term field wheat experiment, which includes plots treated with inorganic fertilizer with and without sulfate, with farmyard manure, and unfertilized plots. Direct isolation of desulfonating rhizobacteria yielded Rhodococcus strains which grew well with a range of sulfonates, and contained the asfAB genes, known to be involved in sulfonate desulfurization by bacteria. Expression of asfA in vitro increased >100-fold during growth of the Rhodococcus isolates with toluenesulfonate as sulfur source, compared with growth with sulfate. By contrast, the closely related Rhodococcus erythropolis and Rhodococcus opacus type strains had no desulfonating activity and did not contain asfA homologues. The overall actinobacterial community structure in wheat rhizospheres was influenced by the sulfur fertilization regime, as shown by specific denaturing gradient gel electrophoresis of PCR amplified 16S rRNA gene fragments, and asfAB clone library analysis identified nine different asfAB genotypes closely affiliated to the Rhodococcus isolates. However, asfAB-based multiplex restriction fragment length polymorphism (RFLP)/terminal-RFLP analysis of wheat rhizosphere communities revealed only slight differences between the fertilization regimes, suggesting that the desulfonating Rhodococcus community does not specifically respond to changes in sulfate supply.

The role of Variovorax and other Comamonadaceae in sulfur transformations by microbial wheat rhizosphere communities exposed to different sulfur fertilization regimes.

Sulfonates are a key component of the sulfur present in agricultural soils. Their mobilization as part of the soil sulfur cycle is mediated by rhizobacteria, and involves the oxidoreductase AsfA. In this study, the effect of fertilization regime on rhizosphere bacterial asfA distribution was examined at the Broadbalk long-term wheat experiment, Rothamsted, UK, which was established in 1843, and has included a sulfur-free treatment since 2001. Direct isolation of desulfonating rhizobacteria from the wheat rhizospheres led to the identification of several Variovorax and Polaromonas strains, all of which contained the asfA gene. Rhizosphere DNA was isolated from wheat rhizospheres in plots fertilized with inorganic fertilizer with and without sulfur, with farmyard manure or from unfertilized plots. Genetic profiling of 16S rRNA gene fragments [denaturing gradient gel electrophoresis (DGGE)] from the wheat rhizospheres revealed that the level of inorganic sulfate in the inorganic fertilizer was correlated with changes in the general bacterial community structure and the betaproteobacterial community structure in particular. Community analysis at the functional gene level (asfA) showed that 40% of clones in asfAB clone libraries were affiliated to the genus Variovorax. Analysis of asfAB-based terminal restriction fragment length polymorphism (T-RFLP) fingerprints showed considerable differences between sulfate-free treatments and those where sulfate was applied. The results suggest the occurrence of desulfonating bacterial communities that are specific to the fertilization regime chosen and that arylsulfonates play an important role in rhizobacterial sulfur nutrition.