Mette Haubjerg Nicolaisen

Microbial plant growth promoters often perform inconsistently under natural soil conditions most likely due to their poor establishment after inoculation. Here, we conducted a growth chamber pot experiment with maize using pre-activated inoculum of the P-solubilizing fungus Penicillium aculeatum to facilitate its establishment in a natural non-sterile agricultural soil. Hence, P. aculeatum was preincubated for one week in soil amended with cellulose as a C source and with different P sources, i.e. sewage sludge ash, Ca ₃ (PO ₄ ) ₂ , and no P fertilization as control. Control treatments of C and P sources without P. aculeatum were also included. The pre-activated inocula were mixed into natural agricultural soil (1:40 w/w), maize seeds were sown, and plants were grown for 42 days. Measured variables included shoot and root dry weight, nutrient content, and root colonization by arbuscular mycorrhizal fungi (AMF) and root infection with Microdochium bolleyi . Overall, P. aculeatum inoculation enhanced maize growth, nutrient content and AMF root colonization, while reduced root infection with M. bolleyi , independent of P source used. Both P sources increased maize growth and nutrient content, though sewage sludge ash showed stronger effect than Ca ₃ (PO ₄ ) ₂ . None of the P sources affected AMF root colonization, but sewage sludge ash decreased M. bolleyi root infection and increased the population density of P. aculeatum . In conclusion, inoculation with pre-activated P. aculeatum (with cellulose and P sources) enhances maize growth and nutrient uptake, and is associated with increased AMF root colonization and reduced root infection by M. bolleyi .

Growing knowledge on plant microbiomes demonstrates the contribution of the host plant during microbiome assembly, especially under stress conditions commonly threatening crops. To dissect the influence of a plant on its microbiome, repeated cycling of microbiomes can be utilized to enhance functional properties in the enriched microbial communities. We used such a successive cultivation approach for wheat (Triticum aestivum) microbiome under drought conditions and selected lineages for drought resilience and susceptibility, with and without enriching the starting community with a library of bacterial isolates obtained from wheat. Significant differences in the rhizosphere microbiome between selection regimes were confirmed through 16S rRNA gene amplicon sequencing. Notably, replicate lineages of each selection regime showed convergence to similar microbiomes. Specific genera were abundant depending on the selection regimes; Stenotrophomonas under drought resilience, while Rahnella under drought conditions when the strain library was added initially. Applying Stenotrophomonas or Rahnella as single inoculum did not improve drought resilience in wheat. We hypothesize that complex microbiome dynamics take place during successive cultivation, which underscores the importance of considering complex plant-microbiome systems for studying plant stress resilience. Successive cultivation remains a valuable approach for observing rhizosphere microbiome changes under different conditions.

Background: Saponins, a diverse group of glycosylated triterpenoid and steroid compounds produced by plants, exhibit potent insecticidal activity and are promising candidates for sustainable pest management. However, their potential persistence in soil raises concerns about ecological impacts, highlighting the need to understand microbial degradation rates and pathways of these compounds. Results: This study reports the isolation of Arthrobacter sp. α11c, and its ability of fully degrading the two hederagenin-based pentacyclic triterpenoid saponins, α-hederin and hederacoside C, as well as the corresponding sapogenin hederagenin. All three compounds were fully metabolized within six hours when provided as carbon source. Fractionation experiments confirmed intracellular uptake and degradation of the saponins. Genome analysis of Arthrobacter α11c revealed multiple putative glycosidase genes. Culturing Arthrobacter α11c with α-hederin, hederacoside C, and the corresponding sapogenin hederagenin did, however, not induce glucosidase activity of the strain. Comparative genomics between Arthrobacter α11c and Arthrobacter sp. α12b, a closely related strain unable to degrade saponins, revealed two unique glycosidase gene clusters in Arthrobacter α11c, suggesting a role in adaptation to saponin metabolism. Conclusions: These findings enhance our understanding of bacterial degradation of plant triterpenoid saponins and provide a foundation for evaluating their environmental fate. This knowledge supports the safe and sustainable use of saponin-based biopesticides in agriculture by identifying microbial partners that contribute to their breakdown in soil ecosystems.

The root microbiome is important for plant development. The impact of the root microbiome is the result of multiple complex interactions among microorganisms, the plant and the environment. This complexity can be reduced by designing synthetic bacterial communities (SynComs) consisting of bacteria isolated from the roots, making it possible to study these interactions. However, the translational power from SynCom experiments to explain field observations is still very low, which demonstrates the need for development of SynComs that colonize plants comparable to what is observed in the field. Hence, we developed a SynCom consisting of 13 different strains from 13 genera with varying phenotypes originating from the roots of winter wheat (Triticum aestivum cv. Sheriff). The SynCom was inoculated into gamma-irradiated soil prior to sowing and community assembly was determined over 4 weeks using 16S rRNA amplicon sequencing. Winter wheat plants inoculated with the developed SynCom grew comparably to plants inoculated with a natural community (NatCom) obtained from a soil solution over the 4-week period. Furthermore, the temporal dynamics of the majority of the SynCom strains mimicked the development in relative abundance of their respective genera in field grown winter wheat of the same cultivar. However, this could not be translated to a different cultivar (Heerup). Our results demonstrate how SynComs inoculated into gamma-irradiated soil can provide a promising framework to study root microbiome assembly and relate the findings to field observations. At the same time, it highlights the plant-genotype specific impact on community assembly.

Plant biologicals are a widely used yet loosely defined term that generally refers to products intended to enhance plant growth, productivity, and protection against pests and diseases. Many, such as microbial inoculants, biostimulants, and plant-derived compounds, also influence microbial communities and processes. The functionality has been recognised for decades, but consistent field-level benefits remain difficult to demonstrate. Advances in formulation and monitoring now offer renewed opportunities to apply biologicals more effectively as tools for managing soil biological processes. They are increasingly recognised for their capacity to influence microbial diversity, stimulate enzymatic activity, and promote nutrient cycling—processes central to soil health. Broader adoption, however, is constrained by unclear definitions of soil health-related effects, lack of harmonised indicators, inconsistent field-testing, and regulatory frameworks misaligned with the characteristics of biological inputs. Drawing on insights from the 2025 Plant Biologicals Network (PBN) workshop on soil health, the opinion paper reviews the current evidence base and key research gaps, highlighting that efficacy remains highly context-dependent and long-term legacy effects are poorly understood. We emphasise the need for emerging technologies and digital tools, including molecular diagnostics, biosensors, and machine learning approaches, that can provide scalable, high-resolution insights into microbial dynamics and soil processes. Finally, we outline a coordinated research and innovation agenda that prioritises harmonised indicators, long-term comparative field measurements, and incentive-aligned frameworks. By integrating scientific, technological, and institutional advances, plant biologicals can evolve from crop-enhancing inputs into credible and scalable instruments for managing soil processes within sustainable soil health strategies.

Secondary metabolites are bioactive compounds, diverse in structure, with versatile ecological functions, including key roles in mediating interactions between microorganisms and plants. Importantly, these compounds can promote the colonization of plant surfaces, such as roots, or modulate root exudates to enhance microbial recruitment and establishment. However, owing to the vast diversity of secondary metabolites, their importance in plant root colonization—particularly under stress conditions, such as drought—remains unclear. To determine the involvement of some of these secondary metabolites in root colonization, we used amplicon sequencing targeting the adenylation domain of the non-ribosomal peptide synthases (NRPSs) and the 16S rRNA gene from the rhizoplane of wheat grown in soil under normal and drought stress conditions. Results showed that drought transiently affected the bacterial community composition and the NRPS composition in the rhizoplane. We observed that drought selected for distinct groups of siderophores from different taxonomical groups, enriching for Streptomyces and depleting Pseudomonas siderophores. In addition, drought enriched Pseudomonas -derived NRPS genes encoding viscosin, a cyclic lipopeptide with biosurfactant properties, indicating that compounds linked to motility and colonization provide a competitive advantage during rhizoplane colonization under drought stress conditions. This observation was experimentally confirmed using the viscosin-producing P. fluorescens SBW25 and its viscosin-deficient mutant. A higher abundance of SBW25 colonized the roots under drought stress conditions compared to the viscosin-deficient mutant. In summary, our work demonstrates the potential for amplicon sequencing of NRPS genes, coupled with in planta experiments, to elucidate the importance of secondary metabolites in root colonization. IMPORTANCE: To harness beneficial plant–microbe interactions for improved plant resilience, we need to advance our understanding of key factors required for successful root colonization. Bacterial-produced secondary metabolites are important in plant–microbe interactions; thus, targeting these genes generates new knowledge that is essential for leveraging bacteria for sustainable agriculture. We used amplicon sequencing of the NRPS A domain on the rhizoplane of wheat exposed to drought stress to identify important secondary metabolites in plant–microbe interactions during drought. We show that the siderophores respond differently to drought stress depending on taxonomic affiliation and that the potential to synthesize viscosin increases root colonization. Importantly, this study demonstrates the potential of amplicon sequencing of NRPS genes to reveal specific secondary metabolites involved in root colonization, particularly in relation to drought stress, and highlights how the resolution provided by this approach can link specific compounds to a specific stress condition in a soil system.

The root microbiome is important for plant development. The impact of the root microbiome is the sum of multiple complex interactions among microorganisms, the plant and the environment. This complexity can be reduced by designing synthetic bacterial communities (SynComs) consisting of bacteria isolated from the roots, making it possible to study these interactions. However, the translational power from SynCom experiments to explain field observations is still very low, which demonstrates the need for development of SynComs that colonize plants comparable to what is observed in the field. Hence, we developed a SynCom consisting of 13 different strains from 13 genera with varying phenotypes originating from the roots of winter wheat ( Triticum aestivum cv. Sheriff). The SynCom was inoculated into gamma-irradiated soil prior to sowing and community assembly was determined over 4 weeks using 16S rRNA amplicon sequencing. The SynCom supported growth of winter wheat over a 4-week period and developed in a comparable manner to a more diverse natural community (NatCom) obtained from a soil solution. Furthermore, the temporal dynamics of the majority of the SynCom strains mimicked the development in relative abundance of their respective genera in field grown winter wheat of similar cultivar. However, this could not be translated to a different cultivar (Herup). Our results demonstrate how SynComs inoculated into gamma-irradiated soil can provide a framework for bridging the gap between greenhouse and lab experiments and field observations. At the same time it highlights the plant-genotype specific impact on community assembly.

Growing knowledge on plant microbiomes demonstrates the contribution of the host plant during microbiome assembly, especially under stress conditions commonly threatening crops. To dissect the influence of a plant on its microbiome, repeated cycling of microbiomes can be utilized to enhance functional properties in the enriched microbial communities. We used such a successive cultivation approach for wheat ( Triticum aestivum ) microbiome under drought conditions and selected lineages for drought resilience and susceptibility, with and without enriching the starting community with a wheat isolate library. Significant differences in the rhizosphere microbiome between selection regimes were confirmed through 16S rRNA gene amplicon sequencing. Notably, replicate lineages of each selection regime showed convergence to similar microbiomes. Specific genera were abundant depending on the selection regimes; Stenotrophomonas under drought resilience, while Rahnella under drought conditions when the strain library was added initially. Applying Stenotrophomonas or Rahnella as single inoculum did not improve drought resilience in wheat. We hypothesize that complex microbiome dynamics take place during successive cultivation, which underscores the importance of considering complex plant-microbiome systems for studying plant stress resilience. Successive cultivation remains a valuable approach for observing rhizosphere microbiome changes under different conditions.

Understanding how crops recruit bacterial and fungal partners for phosphorus (P) nutrition remains incomplete, particularly regarding temporal shifts between P-cycling bacteria and arbuscular mycorrhizal fungi (AMF). We investigated these dynamics using the wheat root hair mutant short root hair 1 (srh1), which is impaired in root hair elongation and may be more dependent on microbial P acquisition compared to the wildtype. We profiled the P-cycling bacterial microbiome and AMF colonization across development and at booting under P-depleted conditions. Before AMF establishment, the root hair mutant rhizosphere was enriched in bacterial organic P-mineralization genes. After AMF colonization increased in both genotypes, this enrichment disappeared, indicating a temporal shift from bacterial to fungal P acquisition. Growing the root hair mutant in P-depleted conditions to booting, when P uptake is highest in wheat, did not induce the mutant to recruit its bacterial P-cycling microbiome. Instead, AMF arbuscule formation was enriched. Collectively, our results reveal a temporal transition in wheat from bacterial to AMF-mediated P nutrition and highlight the compensatory role of the microbiome in plants lacking root hair elongation.

Despite advances in the operation of recirculating aquaculture systems (RAS), accumulation of earthy-muddy off-flavors in the fish remains a potential risk. Myxobacteria (Myxococcota) are reported to be among the most abundant geosmin synthase-harboring groups in RAS, but previous isolation attempts have been unsuccessful, limiting the knowledge of their role in off-flavor production. For the first time, we successfully isolated two geosmin-producing myxobacteria from RAS: Myxococcus virescens AT3 and Corallococcus exiguus AT4. Cell-specific geosmin production varied with the nutrient content in different media but was highest in a low-nutrient medium and when cultivated in water from RAS. Cultivation in RAS water also stimulated the production of other volatile organic compounds (VOCs). Newly identified potential off-flavor compounds included 4-methyl-2-heptanone (“forest” odor), 3-methyl-1-butanol (“medicinal” and “chemical”), and a presumptive sesquiterpenoid described as “musty,” “earthy,” and “flowery.” The previously known off-flavor compound dimethyl sulfide was also detected. Myxobacteria have previously been proposed as keystone bacteria in the environment due to their predatory lifestyle. In predation assays using isolated bacteria from RAS, M. virescens AT3 and C. exiguus AT4 could successfully feed on 15 of 16 tested strains, suggesting a large influence on the biology of RAS microbiomes. The combination of predatory behavior and potent production of geosmin and other VOCs underscores the ecological and sensory impact of these bacteria in RAS. Understanding their behavior and metabolic outputs is critical to developing strategies for mitigating off-flavors in RAS. IMPORTANCE: Issues with off-flavored fish in recirculating aquaculture systems (RAS) due to the presence of the earthy-musty smelling compounds geosmin and 2-MIB are considered as one of the industry’s most economically significant challenges. Knowledge of conditions that affect off-flavor production is essential information in the development of viable solutions for its mitigation. Little is known about the function of these microbially produced compounds or the conditions that trigger their production, especially in the underexplored myxobacteria. Investigation of natural isolates is crucial to determine the function of the genes involved and their differential expression in response to environmental cues. While myxobacteria in RAS have been previously shown to harbor the geosmin synthase gene in molecular studies, the present study is the first attempt to isolate these bacteria from RAS and quantify their geosmin production under various nutrient conditions. Through cultivation-based methods, we demonstrate their production of both known and novel compounds with earthy attributes.