Investigating the Impact of Tillage and Crop Rotation on the Prevalence of phlD-Carrying Pseudomonas Potentially Involved in Disease Suppression.

Winter oilseed rape (OSR) is becoming an increasingly popular crop in rotations as it provides a cash crop and reduces the incidence of take-all fungal disease (caused by Gaeumannomyces graminis) in subsequent wheat production. The exact mechanism of this inhibition of fungal pathogens is not fully understood; however, the selective recruitment of bacterial groups with the ability to suppress pathogen growth and reproduction is thought to play a role. Here we examine the effect of tillage practice on the proliferation of microbes that possess the phlD gene involved in the production of the antifungal compound 2,4-diacetylphloroglucinol (2,4-DAPG), in the rhizospheres of both winter oilseed rape and winter wheat grown in rotation over a two-year period. The results showed that conservation strip tillage led to a significantly greater phlD gene copy number, both in the soil and in the roots, of oilseed rape and wheat crops, whereas crop rotation of oilseed rape and wheat did not increase the phlD gene copy number in winter wheat.

The Good, the Bad, and the Useable Microbes within the Common Alder (Alnus glutinosa) Microbiome-Potential Bio-Agents to Combat Alder Dieback.

Common Alder (Alnus glutinosa (L.) Gaertn.) is a tree species native to Ireland and Europe with high economic and ecological importance. The presence of Alder has many benefits including the ability to adapt to multiple climate types, as well as aiding in ecosystem restoration due to its colonization capabilities within disturbed soils. However, Alder is susceptible to infection of the root rot pathogen Phytophthora alni, amongst other pathogens associated with this tree species. P. alni has become an issue within the forestry sector as it continues to spread across Europe, infecting Alder plantations, thus affecting their growth and survival and altering ecosystem dynamics. Beneficial microbiota and biocontrol agents play a crucial role in maintaining the health and resilience of plants. Studies have shown that beneficial microbes promote plant growth as well as aid in the protection against pathogens and abiotic stress. Understanding the interactions between A. glutinosa and its microbiota, both beneficial and pathogenic, is essential for developing integrated management strategies to mitigate the impact of P. alni and maintain the health of Alder trees. This review is focused on collating the relevant literature associated with Alder, current threats to the species, what is known about its microbial composition, and Common Alder-microbe interactions that have been observed worldwide to date. It also summarizes the beneficial fungi, bacteria, and biocontrol agents, underpinning genetic mechanisms and secondary metabolites identified within the forestry sector in relation to the Alder tree species. In addition, biocontrol mechanisms and microbiome-assisted breeding as well as gaps within research that require further attention are discussed.

Field scale biodegradation of total petroleum hydrocarbons and soil restoration by Ecopiles: microbiological analysis of the process.

Ecopiling is a method for biodegradation of hydrocarbons in soils. It derives from Biopiles, but phytoremediation is added to biostimulation with nitrogen fertilization and bioaugmentation with local bacteria. We have constructed seven Ecopiles with soil heavily polluted with hydrocarbons in Carlow (Ireland). The aim of the study was to analyze changes in the microbial community during ecopiling. In the course of 18 months of remediation, total petroleum hydrocarbons values decreased in 99 and 88% on average for aliphatics and aromatics, respectively, indicating a successful biodegradation. Community analysis showed that bacterial alfa diversity (Shannon Index), increased with the degradation of hydrocarbons, starting at an average value of 7.59 and ending at an average value of 9.38. Beta-diversity analysis, was performed using Bray-Curtis distances and PCoA ordination, where the two first principal components (PCs) explain the 17 and 14% of the observed variance, respectively. The results show that samples tend to cluster by sampling time instead of by Ecopile. This pattern is supported by the hierarchical clustering analysis, where most samples from the same timepoint clustered together. We used DSeq2 to determine the differential abundance of bacterial populations in Ecopiles at the beginning and the end of the treatment. While TPHs degraders are more abundant at the start of the experiment, these populations are substituted by bacterial populations typical of clean soils by the end of the biodegradation process. Similar results are found for the fungal community, indicating that the microbial community follows a succession along the process. This succession starts with a TPH degraders or tolerant enriched community, and finish with a microbial community typical of clean soils.

Assessment of microplastics in Irish river sediment.

Microplastics (MPs) are environmental pollutants of growing concern, and awareness of MPs pollution in marine and freshwater environments has increased in recent years. However, knowledge of MPs contamination in riverine sediments in Ireland is limited. To address this, we collected and analysed sediment samples from 16 selected sites along the River Barrow. Microplastics were extracted through a density separation method, after which their size, colour, and shape were analysed under a stereo microscope (Optica SZM-2). Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to identify polymer types. A total of 690 MPs were recovered from the 16 sites, with fibres as the dominant MP type. The highest concentration of MPs was 155 MP fibres kg-1 wet sediment found in samples collected from Graiguenamanagh, Co. Kilkenny (GK). The majority of the recovered MPs were polyethylene (PE), polypropylene (PP), nylon, and cellulose acetate (CA) fibres. Overall, this study highlighted the presence of MPs in Irish river sediments and provided a baseline for future studies on MPs pollution. Further research is needed to better understand sources, distribution, and effects of MPs in freshwater ecosystems.

Crop Establishment Practices Are a Driver of the Plant Microbiota in Winter Oilseed Rape (Brassica napus).

Gaining a greater understanding of the plant microbiota and its interactions with its host plant heralds a new era of scientific discovery in agriculture. Different agricultural management practices influence soil microbial populations by changing a soil's physical, chemical and biological properties. However, the impact of these practices on the microbiota associated with economically important crops such as oilseed rape, are still understudied. In this work we investigated the impact of two contrasting crop establishment practices, conventional (plow based) and conservation (strip-tillage) systems, on the microbiota inhabiting different plant microhabitats, namely rhizosphere, root and shoot, of winter oilseed rape under Irish agronomic conditions. Illumina 16S rRNA gene sequence profiling showed that the plant associated microhabitats (root and shoot), are dominated by members of the bacterial phyla Proteobacteria, Actinobacteria and Bacteroidetes. The root and shoot associated bacterial communities displayed markedly distinct profiles as a result of tillage practices. We observed a very limited 'rhizosphere effect' in the root zone of WOSR, i.e., there was little or no increase in bacterial community richness and abundance in the WOSR rhizosphere compared to the bulk soil. The two tillage systems investigated did not appear to lead to any major long term differences on the bulk soil or rhizosphere bacterial communities. Our data suggests that the WOSR root and shoot microbiota can be impacted by management practices and is an important mechanism that could allow us to understand how plants respond to different management practices and environments.

Application of Endophytic Pseudomonas fluorescens and a Bacterial Consortium to Brassica napus Can Increase Plant Height and Biomass under Greenhouse and Field Conditions.

Plant associated bacteria with plant growth promotion (PGP) properties have been proposed for use as environmentally friendly biofertilizers for sustainable agriculture; however, analysis of their efficacy in the field is often limited. In this study, greenhouse and field trials were carried out using individual endophytic Pseudomonas fluorescens strains, the well characterized rhizospheric P. fluorescens F113 and an endophytic microbial consortium of 10 different strains. These bacteria had been previously characterized with respect to their PGP properties in vitro and had been shown to harbor a range of traits associated with PGP including siderophore production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and inorganic phosphate solubilization. In greenhouse experiments individual strains tagged with gfp and Kmr were applied to Brassica napus as a seed coat and were shown to effectively colonize the rhizosphere and root of B. napus and in addition they demonstrated a significant increase in plant biomass compared with the non-inoculated control. In the field experiment, the bacteria (individual and consortium) were spray inoculated to winter oilseed rape B. napus var. Compass which was grown under standard North Western European agronomic conditions. Analysis of the data provides evidence that the application of the live bacterial biofertilizers can enhance aspects of crop development in B. napus at field scale. The field data demonstrated statistically significant increases in crop height, stem/leaf, and pod biomass, particularly, in the case of the consortium inoculated treatment. However, although seed and oil yield were increased in the field in response to inoculation, these data were not statistically significant under the experimental conditions tested. Future field trials will investigate the effectiveness of the inoculants under different agronomic conditions.

Draft Genome Sequence of Three Endophyte Strains of Pseudomonas fluorescens Isolated from Miscanthus giganteus.

We report here the draft genome sequence of three Pseudomonas fluorescens strains (L111, L228, and L321) isolated from Miscanthus giganteus The draft genome analyses uncovered a group of genes involved in the biosynthesis of secondary metabolites and for plant growth promotion.

AFLP analysis of genetic diversity and phylogenetic relationships of Brassica oleracea in Ireland.

Brassica oleracea L. is one of the most economically important vegetable crop species of the genus Brassica L. This species is threatened in Ireland, without any prior reported genetic studies. The use of this species is being very limited due to its imprecise phylogeny and uncompleted genetic characterisation. The main objective of this study was to assess the genetic diversity and phylogenetic relationships of a set of 25 Irish B. oleracea accessions using the powerful amplified fragment length polymorphism (AFLP) technique. A total of 471 fragments were scored across all the 11 AFLP primer sets used, out of which 423 (89.8%) were polymorphic and could differentiate the accessions analysed. The dendrogram showed that cauliflowers were more closely related to cabbages than kales were, and accessions of some cabbage types were distributed among different clusters within cabbage subgroups. Approximately 33.7% of the total genetic variation was found among accessions, and 66.3% of the variation resided within accessions. The total genetic diversity (HT) and the intra-accessional genetic diversity (HS) were 0.251 and 0.156, respectively. This high level of variation demonstrates that the Irish B. oleracea accessions studied should be managed and conserved for future utilisation and exploitation in food and agriculture. In conclusion, this study addressed important phylogenetic questions within this species, and provided a new insight into the inclusion of four accessions of cabbages and kales in future breeding programs for improving varieties. AFLP markers were efficient for assessing genetic diversity and phylogenetic relationships in Irish B. oleracea species.

Genetic diversity and population structure of Brassica oleracea germplasm in Ireland using SSR markers.

The most economically important Brassica oleracea species is endangered in Ireland, with no prior reported genetic characterization studies. This study assesses the genetic diversity, population structure and relationships of B. oleracea germplasm in Ireland using microsatellite (SSRs) markers. A total of 118 individuals from 25 accessions of Irish B. oleracea were genotyped. The SSR loci used revealed a total of 47 alleles. The observed heterozygosity (0.699) was higher than the expected one (0.417). Moreover, the average values of fixation indices (F) were negative, indicating excess of heterozygotes in all accessions. Polymorphic information content (PIC) values of SSR loci ranged from 0.27 to 0.66, with an average of 0.571, and classified 10 loci as informative markers (PIC>0.5) to differentiate among the accessions studied. The genetic differentiation among accessions showed that 27.1% of the total genetic variation was found among accessions, and 72.9% of the variation resided within accessions. The averages of total heterozygosity (H(T)) and intra-accession genetic diversity (H(S)) were 0.577 and 0.442, respectively. Cluster analysis of SSR data distinguished among kale and Brussels sprouts cultivars. This study provided a new insight into the exploitation of the genetically diverse spring cabbages accessions, revealing a high genetic variation, as potential resources for future breeding programs. SSR loci were effective for differentiation among the accessions studied.

Plant growth promotion induced by phosphate solubilizing endophytic Pseudomonas isolates.

The use of plant growth promoting bacterial inoculants as live microbial biofertilizers provides a promising alternative to chemical fertilizers and pesticides. Inorganic phosphate solubilization is one of the major mechanisms of plant growth promotion by plant associated bacteria. This involves bacteria releasing organic acids into the soil which solubilize the phosphate complexes converting them into ortho-phosphate which is available for plant up-take and utilization. The study presented here describes the ability of endophytic bacteria to produce gluconic acid (GA), solubilize insoluble phosphate, and stimulate the growth of Pisum sativum L. plants. This study also describes the genetic systems within three of these endophyte strains thought to be responsible for their effective phosphate solubilizing abilities. The results showed that many of the endophytic strains produced GA (14-169 mM) and have moderate to high phosphate solubilization capacities (~400-1300 mg L(-1)). When inoculated into P. sativum L. plants grown in soil under soluble phosphate limiting conditions, the endophytes that produced medium-high levels of GA displayed beneficial plant growth promotion effects.

Ecopiling: a combined phytoremediation and passive biopiling system for remediating hydrocarbon impacted soils at field scale.

Biopiling is an ex situ bioremediation technology that has been extensively used for remediating a wide range of petrochemical contaminants in soils. Biopiling involves the assembling of contaminated soils into piles and stimulating the biodegrading activity of microbial populations by creating near optimum growth conditions. Phytoremediation is another very successful bioremediation technique and involves the use of plants and their associated microbiomes to degrade, sequester or bio-accumulate pollutants from contaminated soil and water. The objective of this study was to investigate the effectiveness of a combined phytoremediation/biopiling system, termed Ecopiling, to remediate hydrocarbon impacted industrial soil. The large scale project was carried out on a sandy loam, petroleum impacted soil [1613 mg total petroleum hydrocarbons (TPHs) kg(-1) soil]. The contaminated soil was amended with chemical fertilizers, inoculated with TPH degrading bacterial consortia and then used to construct passive biopiles. Finally, a phyto-cap of perennial rye grass (Lolium perenne) and white clover (Trifolium repens) was sown on the soil surface to complete the Ecopile. Monitoring of important physico-chemical parameters was carried out at regular intervals throughout the trial. Two years after construction the TPH levels in the petroleum impacted Ecopiles were below detectable limits in all but one subsample (152 mg TPH kg(-1) soil). The Ecopile system is a multi-factorial bioremediation process involving bio-stimulation, bio-augmentation and phytoremediation. One of the key advantages to this system is the reduced costs of the remediation process, as once constructed, there is little additional cost in terms of labor and maintenance (although the longer process time may incur additional monitoring costs). The other major advantage is that many ecological functions are rapidly restored to the site and the process is esthetically pleasing.

Genome sequence reveals that Pseudomonas fluorescens F113 possesses a large and diverse array of systems for rhizosphere function and host interaction.

BACKGROUND: Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) isolated from the sugar-beet rhizosphere. This bacterium has been extensively studied as a model strain for genetic regulation of secondary metabolite production in P. fluorescens, as a candidate biocontrol agent against phytopathogens, and as a heterologous host for expression of genes with biotechnological application. The F113 genome sequence and annotation has been recently reported. RESULTS: Comparative analysis of 50 genome sequences of strains belonging to the P. fluorescens group has revealed the existence of five distinct subgroups. F113 belongs to subgroup I, which is mostly composed of strains classified as P. brassicacearum. The core genome of these five strains is highly conserved and represents approximately 76% of the protein-coding genes in any given genome. Despite this strong conservation, F113 also contains a large number of unique protein-coding genes that encode traits potentially involved in the rhizocompetence of this strain. These features include protein coding genes required for denitrification, diterpenoids catabolism, motility and chemotaxis, protein secretion and production of antimicrobial compounds and insect toxins. CONCLUSIONS: The genome of P. fluorescens F113 is composed of numerous protein-coding genes, not usually found together in previously sequenced genomes, which are potentially decisive during the colonisation of the rhizosphere and/or interaction with other soil organisms. This includes genes encoding proteins involved in the production of a second flagellar apparatus, the use of abietic acid as a growth substrate, the complete denitrification pathway, the possible production of a macrolide antibiotic and the assembly of multiple protein secretion systems.

Genome sequence of the biocontrol strain Pseudomonas fluorescens F113.

Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) that has biocontrol activity against fungal plant pathogens and is a model for rhizosphere colonization. Here, we present its complete genome sequence, which shows that besides a core genome very similar to those of other strains sequenced within this species, F113 possesses a wide array of genes encoding specialized functions for thriving in the rhizosphere and interacting with eukaryotic organisms.

Genetically modified Pseudomonas biosensing biodegraders to detect PCB and chlorobenzoate bioavailability and biodegradation in contaminated soils.

Whole cell microbial biosensors offer excellent possibilities for assaying the complex nature of the bioavailable and bioaccessible fraction of pollutants in contaminated soils, which currently cannot be easily addressed. This paper describes the application and evaluation of three microbial biosensor strains designed to detect the bioavailability and biodegradation of PCBs (and end-products) in contaminated soils and sediments. Polychlorinated biphenyls (PCBs) are considered to be one of the most wide spread, hazardous and persistent pollutants. Herein we describe that there was a positive correlation between the PCB levels within the samples and the percentage of biosensor cells that were expressing their reporter gene; gfp. Immobilisation of the biosensors in calcium alginate beads allowed easy and accurate detection of the biosensor strains in contaminated soil and sludge samples. The biosensors also showed that PCB degradation activity was occurring at a much greater level in Pea inoculated planted soil compared to inoculated unplanted soil indicating rhizoremediation (the removal of pollutants by plant root associated microbes) shows considerable promise as a solution for removing organic xenobiotics from the environment.

Whole-cell fluorescent biosensors for bioavailability and biodegradation of polychlorinated biphenyls.

Whole-cell microbial biosensors are one of the newest molecular tools used in environmental monitoring. Such biosensors are constructed through fusing a reporter gene such as lux, gfp or lacZ, to a responsive promoter. There have been many reports of the applications of biosensors, particularly their use in assaying pollutant toxicity and bioavailability. This paper reviews the basic concepts behind the construction of whole-cell microbial biosensors for pollutant monitoring, and describes the applications of two such biosensors for detecting the bioavailability and biodegradation of polychlorinated biphenyls (PCBs).

Bacterial endophyte-mediated naphthalene phytoprotection and phytoremediation.

Polyaromatic hydrocarbons (PAHs) are major and recalcitrant pollutants of the environment and their removal presents a significant problem. Phytoremediation has shown much promise in PAH removal from contaminated soil, but may be inhibited because the plant experiences phytotoxic effects from low-molecular-weight PAHs such as naphthalene. This paper describes the construction of a naphthalene-degrading endophytic strain designated Pseudomonas putida VM1441(pNAH7). This strain was found to be an efficient colonizer of plants, colonizing both the rhizosphere and interior root tissues. The inoculation of plants with P. putida VM1441(pNAH7) resulted in the protection of the host plant from the phytotoxic effects of naphthalene. When inoculated plants were exposed to naphthalene, both seed germination and plant transpiration rates were higher than those of the uninoculated controls. The inoculation of plants with this strain also facilitated higher (40%) naphthalene degradation rates compared with uninoculated plants in artificially contaminated soil.

Bacterial endophytes: recent developments and applications.

Endophytic bacteria have been found in virtually every plant studied, where they colonize the internal tissues of their host plant and can form a range of different relationships including symbiotic, mutualistic, commensalistic and trophobiotic. Most endophytes appear to originate from the rhizosphere or phyllosphere; however, some may be transmitted through the seed. Endophytic bacteria can promote plant growth and yield and can act as biocontrol agents. Endophytes can also be beneficial to their host by producing a range of natural products that could be harnessed for potential use in medicine, agriculture or industry. In addition, it has been shown that they have the potential to remove soil contaminants by enhancing phytoremediation and may play a role in soil fertility through phosphate solubilization and nitrogen fixation. There is increasing interest in developing the potential biotechnological applications of endophytes for improving phytoremediation and the sustainable production of nonfood crops for biomass and biofuel production.

Bacterial endophyte-enhanced phytoremediation of the organochlorine herbicide 2,4-dichlorophenoxyacetic acid.

2,4-Dichlorophenoxyacetic acid is a selective systemic herbicide for the control of broad-leaved weeds, which is widely used throughout the world. The persistence of its residues and its potential to migrate in the soil make it necessary to reduce its concentrations in contaminated soil and groundwater. The nature of this compound makes it particularly toxic to the broad-leaved plants, such as the poplar (Populus) and willow (Salix), which are often used in phytoremediation projects. We describe the inoculation of a model plant, the pea (Pisum sativum), with a genetically tagged bacterial endophyte that naturally possesses the ability to degrade 2,4-dichlorophenoxyacetic acid. The results showed that this strain actively colonized inoculated plants internally (and in the rhizosphere). Inoculated plants showed a higher capacity for 2,4-dichlorophenoxyacetic acid removal from soil and showed no 2,4-dichlorophenoxyacetic acid accumulation in their aerial tissues. This demonstrates the usefulness of bacterial endophytes to enhance the phytoremediation of herbicide-contaminated substrates and reduce levels of toxic herbicide residues in crop plants.

Colonisation of poplar trees by gfp expressing bacterial endophytes.

With the exception of nitrogen fixing bacteria, there is little known about the colonisation patterns or population sizes of bacterial endophytes in deciduous trees. This study describes the isolation, identification, construction and re-colonisation patterns of three green fluorescent protein(gfp):kanamycin(R) labelled bacterial endophytes when re-introduced into poplar trees, their original host plant. Two of these endophytes showed considerable colonisation in the roots and stems of inoculated plants. gfp expressing cells of all three strains were observed to colonise the xylem tissue of the root. All three strains proved to be efficient rhizosphere colonisers, supporting the theory that the rhizosphere can serve as a source of bacterial endophytes.