Bram Beckers

Potential of willow and its genetically engineered associated bacteria to remediate mixed Cd and toluene contamination

PurposeThe purpose of this study was to investigate if bacteria with beneficial properties that were isolated from willow growing on a metal-contaminated site can be further equipped with genes coding for a specific degradation pathway to finally obtain transconjugants that can be inoculated in willow to improve phytoremediation efficiency of mixed contaminations.Materials and methodsCultivable rhizosphere bacteria and root endophytes were isolated from willow (cv. Tora) growing on a metal-contaminated soil. All isolated strains were tested for their metal resistance and potential to promote plant growth. The two most promising strains were selected and were equipped with the pTOM plasmid coding for toluene degradation. Both transconjugants were inoculated separately and combined in willow cuttings exposed to mixed Cd–toluene contamination, and their effect on phytotoxicity, Cd uptake, and toluene evapotranspiration was evaluated.Results and discussionMany of the isolated strains tested positive for the production of siderophores, organic acids, and indole acetic acid (IAA) and showed increased Cd resistance. The Cd-resistant, siderophore-producing rhizosphere strain Burkholderia sp. HU001 and the Cd-resistant root endophyte Pseudomonas sp. HU002, able to produce siderophores, organic acids, and IAA, were selected as receptors for conjugation with the toluene-degrading Burkholderia vietnamiensis BU61 as a donor of the pTOM-TCE plasmid. Although inoculation with the individual transconjugant strains had no effect on plant growth and negatively affected Cd uptake, their combined inoculation resulted in an increased shoot biomass upon Cd–toluene exposure did not affect Cd uptake and strongly reduced evapotranspiration of toluene to the atmosphere.ConclusionsIn this study, inoculation of willow with a consortium of plant-associated bacteria equipped with the appropriate characteristics resulted in an improved phytoremediation of a mixed Cd–toluene contamination: the degradation of toluene was improved leading to a decreased toxicity and evapotranspiration, while Cd uptake and translocation were not affected.

Lignin engineering in field-grown poplar trees affects the endosphere bacterial microbiome

Significance The composition of bacterial populations in the human digestive tract is known to affect our well-being, to influence our ability to overcome diseases, and to be tightly linked with the host genotype. In contrast, the extent to which variation in the plant genotype affects the associated bacteria and, hence, plant health and productivity remains unknown, particularly of field-grown plants. Genetically modified poplars, down-regulated for cinnamoyl-CoA reductase, substantially influence the microbiome of the plant endosphere without perceptible impact on the rhizosphere microbiota. Unraveling the host genotype-dependent plant–microbe associations is crucial to comprehend the effects of engineering the plant metabolic pathway and possibly to exploit the eukaryote–prokaryote associations in phytoremediation applications, sustainable crop production, and the production of secondary metabolites. Cinnamoyl-CoA reductase (CCR), an enzyme central to the lignin biosynthetic pathway, represents a promising biotechnological target to reduce lignin levels and to improve the commercial viability of lignocellulosic biomass. However, silencing of the CCR gene results in considerable flux changes of the general and monolignol-specific lignin pathways, ultimately leading to the accumulation of various extractable phenolic compounds in the xylem. Here, we evaluated host genotype-dependent effects of field-grown, CCR-down-regulated poplar trees (Populus tremula × Populus alba) on the bacterial rhizosphere microbiome and the endosphere microbiome, namely the microbiota present in roots, stems, and leaves. Plant-associated bacteria were isolated from all plant compartments by selective isolation and enrichment techniques with specific phenolic carbon sources (such as ferulic acid) that are up-regulated in CCR-deficient poplar trees. The bacterial microbiomes present in the endosphere were highly responsive to the CCR-deficient poplar genotype with remarkably different metabolic capacities and associated community structures compared with the WT trees. In contrast, the rhizosphere microbiome of CCR-deficient and WT poplar trees featured highly overlapping bacterial community structures and metabolic capacities. We demonstrate the host genotype modulation of the plant microbiome by minute genetic variations in the plant genome. Hence, these interactions need to be taken into consideration to understand the full consequences of plant metabolic pathway engineering and its relation with the environment and the intended genetic improvement.

The Potential of the Ni-Resistant TCE-Degrading Pseudomonas putida W619-TCE to Reduce Phytotoxicity and Improve Phytoremediation Efficiency of Poplar Cuttings on A Ni-TCE Co-Contamination.

To examine the potential of Pseudomonas putida W619-TCE to improve phytoremediation of Ni-TCE co-contamination, the effects of inoculation of a Ni-resistant, TCE-degrading root endophyte on Ni-TCE phytotoxicity, Ni uptake and trichloroethylene (TCE) degradation of Ni-TCE-exposed poplar cuttings are evaluated. After inoculation with P. putida W619-TCE, root weight of non-exposed poplar cuttings significantly increased. Further, inoculation induced a mitigation of the Ni-TCE phytotoxicity, which was illustrated by a diminished exposure-induced increase in activity of antioxidative enzymes. Considering phytoremediation efficiency, inoculation with P. putida W619-TCE resulted in a 45% increased Ni uptake in roots as well as a slightly significant reduction in TCE concentration in leaves and TCE evapotranspiration to the atmosphere. These results indicate that endophytes equipped with the appropriate characteristics can assist their host plant to deal with co-contamination of toxic metals and organic contaminants during phytoremediation. Furthermore, as poplar is an excellent plant for biomass production as well as for phytoremediation, the obtained results can be exploited to produce biomass for energy and industrial feedstock applications in a highly productive manner on contaminated land that is not suited for normal agriculture. Exploiting this land for biomass production could contribute to diminish the conflict between food and bioenergy production.

Phytoremediation of Metal Contaminated Soil Using Willow: Exploiting Plant-Associated Bacteria to Improve Biomass Production and Metal Uptake

Short rotation coppice (SRC) of willow and poplar is proposed for economic valorization and concurrently as remediation strategy for metal contaminated land in northeast-Belgium. However, metal phytoextraction appears insufficient to effectuate rapid reduction of soil metal contents. To increase both biomass production and metal accumulation of SRC, two strategies are proposed: (i) in situ selection of the best performing clones and (ii) bioaugmentation of these clones with beneficial plant-associated bacteria. Based on field data, two experimental willow clones, a Salix viminalis and a Salix alba x alba clone, were selected. Compared to the best performing commercial clones, considerable increases in stem metal extraction were achieved (up to 74% for Cd and 91% for Zn). From the selected clones, plant-associated bacteria were isolated and identified. All strains were subsequently screened for their plant growth-promoting and metal uptake enhancing traits. Five strains were selected for a greenhouse inoculation experiment with the selected clones planted in Cd-Zn-Pb contaminated soil. Extraction potential tended to increase after inoculation of S. viminalis plants with a Rahnella sp. strain due to a significantly increased twig biomass. However, although bacterial strains showing beneficial traits in vitro were used for inoculation, increments in extraction potential were not always observed.

Plant-associated bacteria and their role in the success or failure of metal phytoextraction projects: first observations of a field-related experiment.

Phytoextraction has been reported as an economically and ecologically sound alternative for the remediation of metal‐contaminated soils. Willow is a metal phytoextractor of interest because it allows to combine a gradual contaminant removal with production of biomass that can be valorized in different ways. In this work two willow clones growing on a metal‐contaminated site were selected: ‘Belgisch Rood’ (BR) with a moderate metal extraction capacity and ‘Tora’ (TO) with a twice as high metal accumulation. All cultivable bacteria associated with both willow clones were isolated and identified using 16SrDNA ARDRA analysis followed by 16SrDNA sequencing. Further all isolated bacteria were investigated for characteristics that might promote plant growth (production of siderophores, organic acids and indol acetic acid) and for their metal resistance. The genotypic and phenotypic characterization of the isolated bacteria showed that the TO endophytic bacterial population is more diverse and contains a higher percentage of metal‐resistant plant growth promoting bacteria than the endophytic population associated with BR. We hypothesize that the difference in the metal accumulation capacity between BR and TO clones might be at least partly related to differences in characteristics of their associated bacterial population.

Structural variability and niche differentiation in the rhizosphere and endosphere bacterial microbiome of field-grown poplar trees

BackgroundThe plant microbiome represents one of the key determinants of plant health and productivity by providing a plethora of functional capacities such as access to low-abundance nutrients, suppression of phytopathogens, and resistance to biotic and/or abiotic stressors. However, a robust understanding of the structural composition of the bacterial microbiome present in different plant microenvironments and especially the relationship between below-ground and above-ground communities has remained elusive. In this work, we addressed hypotheses regarding microbiome niche differentiation and structural stability of the bacterial communities within different ecological plant niches.MethodsWe sampled the rhizosphere soil, root, stem, and leaf endosphere of field-grown poplar trees (Populus tremula × Populus alba) and applied 16S rRNA amplicon pyrosequencing to unravel the bacterial communities associated with the different plant habitats.ResultsWe found that the structural variability of rhizosphere microbiomes in field-grown poplar trees (P. tremula × P. alba) is much lower than that of the endosphere microbiomes. Furthermore, our data not only confirm microbiome niche differentiation reports at the rhizosphere soil–root interface but also clearly show additional fine-tuning and adaptation of the endosphere microbiome in the stem and leaf compartment. Each plant compartment represents an unique ecological niche for the bacterial communities. Finally, we identified the core bacterial microbiome associated with the different ecological niches of Populus.ConclusionsUnderstanding the complex host–microbe interactions of Populus could provide the basis for the exploitation of the eukaryote–prokaryote associations in phytoremediation applications, sustainable crop production (bio-energy efficiency), and/or the production of secondary metabolites.

Cadmium-induced and trans-generational changes in the cultivable and total seed endophytic community of Arabidopsis thaliana.

Trans-generational adaptation is important to respond rapidly to environmental challenges and increase overall plant fitness. Besides well-known mechanisms such as epigenetic modifications, vertically transmitted endophytic bacteria might contribute to this process. The cultivable and total endophytic communities of several generations of Arabidopsis thaliana seeds harvested from plants exposed to cadmium (Cd) or not exposed were investigated. The diversity and richness of the seed endophytic community decreased with an increasing number of generations. Aeromicrobium and Pseudonocardia were identified as indicator species in seeds from Cd-exposed plants, while Rhizobium was abundantly present in both seed types. Remarkably, Rhizobium was the only genus that was consistently detected in seeds of all generations, which suggests that the phenotypic characteristics were more important as selection criteria for which bacteria are transferred to the next plant generation than the actual genera. Production of IAA was an important trait for endophytes from both seed types, while ACC deaminase activity and Cd tolerance were mainly associated with seed endophytes from Cd-exposed plants. Understanding how different factors influence the seed endophytic community can help us to improve seed quality and plant growth through different biotechnological applications.

Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies

Next-generation sequencing technologies have revolutionized the methods for studying microbial ecology by enabling high-resolution community profiling. However, the use of these technologies in unraveling the plant microbiome remains challenging. Many bacterial 16S rDNA primer pairs also exhibit high affinity for non-target DNA such as plastid (mostly chloroplast) DNA and mitochondrial DNA. Therefore, we experimentally tested a series of commonly used primers for the analysis of plant-associated bacterial communities using 454 pyrosequencing. We evaluated the performance of all selected primer pairs in the study of the bacterial microbiomes present in the rhizosphere soil, root, stem and leaf endosphere of field-grown poplar trees (Populus tremula × Populus alba) based on (a) co-amplification of non-target DNA, (b) low amplification efficiency for pure chloroplast DNA (real-time PCR), (c) high retrieval of bacterial 16S rDNA, (d) high operational taxonomic unit (OTU) richness and Inverse Simpson diversity and (e) taxonomic assignment of reads. Results indicate that experimental evaluation of primers provide valuable information that could contribute in the selection of suitable primer pairs for 16S rDNA metabarcoding studies in plant-microbiota research. Furthermore, we show that primer pair 799F-1391R outperforms all other primer pairs in our study in the elimination of non-target DNA and retrieval of bacterial OTUs.

Community structure and diversity of endophytic bacteria in seeds of three consecutive generations of Crotalaria pumila growing on metal mine residues

AimsWe investigated the possible transgenerational transfer of bacterial seed endophytes across three consecutive seed generations of Crotalaria pumila growing on a metal mining site in Mexico.MethodsSeeds were collected during three successive years in the semi-arid region of Zimapan, Mexico. Total communities of seed endophytes were investigated using DNA extraction from surface sterilized seeds and 454 pyrosequencing of the V5-V7 hypervariable regions of the 16S rRNA gene.ResultsThe communities consisted of an average of 75 operational taxonomic units (OTUs); richness and diversity did not change across years. Methylobacterium, Staphylococcus, Corynebacterium, Propionibacterium and eight other OTUs constituted >60% of the community in each generation. The microbiome was dominated by Methylobacterium (present in >80% of samples). Functions associated with the microbiome were C and N fixation, oxidative phosphorylation and photosynthesis activity.ConclusionsThe bacterial endophytic communities were similar across three consecutive seed generations. Among the core microbiome Methylobacterium strains were the most abundant and they can contribute to nutrient acquisition, plant growth promotion and stress resilience to their host in metal contaminated mine residues. Identification of the seed microbiome of C. pumila may lead to novel and more efficient inoculants for microbe-assisted phytoremediation.

Comparison between cultivated and total bacterial communities associated with Cucurbita pepo using cultivation-dependent techniques and 454 pyrosequencing

Endophytic bacteria often have beneficial effects on their host plants that can be exploited for bioremediation applications but, according to the literature, only 0.001-1% of all endophytic microbes should be cultivable. This study compared the cultivated endophytic communities of the roots and shoots of Cucurbita pepo with the total endophytic communities as determined by cultivation-dependent techniques and 454 pyrosequencing. The ten most abundant taxa of the total communities aligned well with the cultivated taxa; however, the abundance of these taxa in the two communities differed greatly. Enterobacter showed very low presence in the total communities, whereas they were dominantly present in the cultivated communities. Although Rhizobium dominated in total root and shoot communities, it was poorly cultivable and even then only in growth media containing plant extract. Since endophytes likely contribute to plant-growth promotion, cultivated bacterial strains were tested for their plant-growth promoting capabilities, and the results were correlated with their abundance in the total community. Bacillus and Pseudomonas showed promising results when considering cultivability, abundance in the total community and plant-growth promoting capability. This study demonstrated that, although a limited number of bacterial genera were cultivable, current cultivation-dependent techniques may be sufficient for further isolation and inoculation experiments that aim to improve phytoremediation efficiency.