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The provision of probiotics benefits the health of a wide range of organisms, from humans to animals and plants. Probiotics can enhance stress resilience of endangered organisms, many of which are critically threatened by anthropogenic impacts. The use of so-called 'probiotics for wildlife' is a nascent application, and the field needs to reflect on standards for its development, testing, validation, risk assessment, and deployment. Here, we identify the main challenges of this emerging intervention and provide a roadmap to validate the effectiveness of wildlife probiotics. We cover the essential use of inert negative controls in trials and the investigation of the probiotic mechanisms of action. We also suggest alternative microbial therapies that could be tested in parallel with the probiotic application. Our recommendations align approaches used for humans, aquaculture, and plants to the emerging concept and use of probiotics for wildlife.

Certain soils promote crop health because they are pathogen-suppressive (i.e., fungistatic) or disease-suppressive, but the effect of soil management on these properties is not fully understood. Here, we tested the hypothesis that manure could favor fungistasis by screening 26 manured or non-manured wheat fields from Serbia for their ability to control survival/growth of the fungal plant pathogen Fusarium graminearum Fg1. Quantitative PCR showed that the pathogen grew after inoculation in all 26 autoclaved soils. In absence of autoclaving, the pathogen was stable or grew in 16 soils (37 % manured) but declined in the 10 others (70 % manured). For most soils, there was no significant link between soil chemistry and fungistasis, except with Mionica in western/central Serbia. Mionica soils MI2 and MI3, which had received manure, exhibited higher levels of organic matter and potassium compared with soils MI4 and MI5, which had not received manure and were non-fungistatic. Using Mionica soils, we then tested the hypothesis that fungistatic (manured) soils rather than non-fungistatic (non-manured) soils would protect wheat from F. graminearum disease. Indeed, fungistatic soils were suppressive to wheat damping-off. Non-fungistatic soil MI4 was conducive, as expected, but non-fungistatic soil MI5 turned out to be suppressive. Metabarcoding showed that the structure of prokaryotic and fungal rhizosphere communities depended mostly on field location, with a significant effect of F. graminearum inoculation. In conclusion, our findings show that certain farming practices (here, manure amendments) may promote soil fungistasis towards F. graminearum. However, both fungistatic and non-fungistatic soils can be suppressive to F. graminearum disease in wheat, and their differences in rhizosphere microbiota suggest different phytoprotection mechanisms.

Introduction Inoculation of plants with beneficial microorganisms may improve plant performance yet suffers from efficacy variability. A solution might be the combined application of different inoculants as consortium. The objective of the present study was to evaluate the effects of single or combined inoculation of Trichoderma harzianum, strain TGFG411, and a consortium of arbuscular mycorrhizal fungi (AMF) on plant growth, and native microbial communities (here bacteria/archaea, fungi and AMF) in root-associated soil (RAS) and rhizosphere (RH), that is, soil loosely or tightly attached to the roots, respectively. Materials and Methods A greenhouse experiment was carried out with non-sterile agricultural soil and the model crop maize, which was single inoculated with either TGFG411 or AMF or received a combined inoculation of TGFG411 + AMF. Control plants received only water. Seven weeks after the second AMF inoculation, the plant growth promotion capacity of the inoculants was measured based on shoot and root parameters. Furthermore, RAS and RH microbiota (fungi including AMF, bacteria and archaea) were assessed via a combination of different cultivation-dependent, microscopic and DNA-based methods. Results After 7 weeks of maize growth, both single and combined inoculation of AMF and TGFG411 enhanced shoot dry weight and led to a significant reduction in root biomass. The TGFG411 strain successfully established in the soil. However, no definite evidence for the establishment of the inoculated AMF was found. Single or combined inoculation of TGFG411 and AMF modified the composition of total bacterial in the RH, whereas modulated total fungal communities in the RAS. Conclusion The combined inoculation did not result in a significant improvement of plant performance compared with single inoculation likely due to optimal nutrient supply. However, samples receiving the combined inoculation exhibited a distinct modulation of the native RAS/RH microbiota, which may inf

Mixed systems of grain and livestock production based on pasture-crop rotations are a promising strategy to promote agriculture resilience and allow an ecological intensification of agriculture yet little is known about underlying processes in soil. To test the hypothesis that pasture-crop rotations preserve soil structure and select for beneficial soil and rhizosphere microbiota, supporting soil health and grain production, a long-term field experiment under no-tillage was studied. The experiment evaluated a gradient of land use intensities and vegetation diversities, from highly intensive continuous cropping to the least intensive system i.e. a nearby natural grassland, with two intermediate land use intensities i.e. short pasture-crop rotation and long pasture-crop rotation. Soil health was assessed based on soil physicochemical properties, microbial (Bacteria/Archaea and Fungi) community diversity and composition and oat performance. Pasture-crop rotations preserved soil bulk density and larger aggregates better than continuous cropping. High-throughput amplicon sequencing of 16S rRNA gene and ITS fragments revealed that the pasture-crop rotations fostered taxa that are associated with soil structure maintenance and selected potential plant-beneficial bacterial genera in the oat rhizosphere (i.e. Bosea, Devosia and Microbacterium), that may have contributed to the observed increase in N uptake, N accumulation and biomass in oat. In summary, this study shows that pasture-crop rotations are an ecologically sustainable alternative to continuous cropping in the Uruguayan Pampa biome.

Approaches to rapidly collecting global biodiversity data are increasingly important, but biodiversity blind spots persist. We organized a three-day Datathon event to improve the openness of local biodiversity data and facilitate data reuse by local researchers. The first Datathon, organized among microbial ecologists in Uruguay and Argentina assembled the largest microbiome dataset in the region to date and formed collaborative consortia for microbiome data synthesis.

Aims Different drivers are known to shape rhizosphere microbiome assembly. How soil texture (Texture) and presence or lack of root hairs (Root Hair) of plants affect the rhizosphere microbiome assembly and soil potential extracellular enzyme activities (EEA) at defined rooting depth (Depth) is still a knowledge gap. We investigated effects of these drivers on microbial assembly in rhizosphere and on potential EEA in root-affected soil of maize.   Methods Samples were taken from three depths of root hair defective mutant rth3 and wild-type WT maize planted on loam and sand in soil columns after 22 days. Rhizosphere bacterial, archaeal, fungal and cercozoan communities were analysed by sequencing of 16S rRNA gene, ITS and 18S rRNA gene fragments. Soil potential EEA of ß-glucosidase, acid phosphatase and chitinase were estimated using fluorogenic substrates.   Results The bacterial, archaeal and cercozoan alpha- and beta-diversities were significantly and strongly altered by Texture, followed by Depth and Root Hair. Texture and Depth had a small impact on fungal assembly, and only fungal beta-diversity was significantly affected. Significant impacts by Depth and Root Hair on beta-diversity and relative abundances at taxonomic levels of bacteria, archaea, fungi and cercozoa were dependent on Texture. Likewise, the patterns of potential EEA followed the trends of microbial communities, and the potential EEA correlated with the relative abundances of several taxa.   Conclusions Texture was the strongest driver of rhizosphere microbiome and of soil potential EEA, followed by Depth and Root Hair, similarly to findings in maize root architecture and plant gene expression studies.

In a field experiment we investigated the influence of the environmental filters soil type (i.e. three contrasting soils) and plant species (i.e. lettuce and potato) identity on rhizosphere community assembly of Cercozoa, a dominant group of mostly bacterivorous soil protists. Plant species (14%) and rhizosphere origin (vs bulk soil) with 13%, together explained four times more variation in cercozoan beta diversity than the three soil types (7% explained variation). Our results clearly confirm the existence of plant species-specific protist communities. Network analyses of bacteria-Cercozoa rhizosphere communities identified scale-free small world topologies, indicating mechanisms of self-organization. While the assembly of rhizosphere bacterial communities is bottom-up controlled through the resource supply from root (secondary) metabolites, our results support the hypothesis that the net effect may depend on the strength of top-down control by protist grazers. Since grazing of protists has a strong impact on the composition and functioning of bacteria communities, protists expand the repertoire of plant genes by functional traits, and should be considered as ‘protist microbiomes’ in analogy to ‘bacterial microbiomes’.

A soil column split-root experiment was designed to investigate the ability of apple replant disease (ARD)-causing agents to spread in soil. ‘M26’ apple rootstocks grew into a top layer of Control soil, followed by a barrier-free split-soil layer (Control soil/ARD soil). We observed a severely reduced root growth, concomitant with enhanced gene expression of phytoalexin biosynthetic genes and phytoalexin content in roots from ARD soil, indicating a pronounced local plant defense response. Amplicon sequencing (bacteria, archaea, fungi) revealed local shifts in diversity and composition of microorganisms in the rhizoplane of roots from ARD soil. An enrichment of operational taxonomic units affiliated to potential ARD fungal pathogens (Ilyonectria and Nectria sp.) and bacteria frequently associated with ARD (Streptomyces, Variovorax) was noted. In conclusion, our integrated study supports the idea of ARD being local and not spreading into surrounding soil, as only the roots in ARD soil were affected in terms of growth, phytoalexin biosynthetic gene expression, phytoalexin production and altered microbiome structure. This study further reinforces the microbiological nature of ARD, being likely triggered by a disturbed soil microbiome enriched with low mobility of the ARD-causing agents that induce a strong plant defense and rhizoplane microbiome dysbiosis, concurring with root damage.

Preservation of the phytostimulatory functions of plant growth-promoting bacteria relies on the adaptation of their community to the rhizosphere environment. Here, an amplicon sequencing approach was implemented to specifically target microorganisms with 1-aminocyclopropane-1-carboxylate deaminase activity, carrying the acdS gene. We stated the hypothesis that the relative phylogenetic distribution of acdS carrying microorganisms is affected by the presence or absence of root hairs, soil type and depth. To this end, a standardized soil column experiment was conducted with maize wild type and root hair defective rth3 mutant in the substrates loam and sand, and harvest was implemented from three depths. Most acdS sequences (99%) were affiliated to Actinobacteria and Proteobacteria, and the strongest influence on the relative abundances of sequences were exerted by the substrate. Variovorax, Acidovorax and Ralstonia sequences dominated in loam, whereas Streptomyces and Agromyces were more abundant in sand. Soil depth caused strong variations in acdS sequence distribution, with differential levels in the relative abundances of acdS sequences affiliated to Tetrasphaera, Amycolatopsis and Streptomyces in loam, but Burkholderia, Paraburkholderia and Variovorax in sand. Maize genotype influenced the distribution of acdS sequences mainly in loam and only in the uppermost depth. Variovorax acdS sequences were more abundant in WT, but Streptomyces, Microbacterium and Modestobacter in rth3 rhizosphere.Substrate and soil depth were strong and plant genotype a further significant single and interacting drivers of acdS carrying microbial community composition in the rhizosphere of maize. This suggests that maize rhizosphere acdS carrying bacterial community establishes according to the environmental constraints, and that root hairs possess a minor but significant impact on acdS carrying bacterial populations.

Non-invasive X-ray computed tomography (XRCT) is increasingly used in rhizosphere research to visualize development of soil–root interfaces in situ. However, exposing living systems to X-rays can potentially impact their processes and metabolites. In order to evaluate these effects, we assessed the responses of rhizosphere processes 1 h and 24 h after a low X-ray exposure (0.81 Gy). Changes in root gene expression patterns occurred 1 h after exposure with down-regulation of cell wall-, lipid metabolism-, and cell stress-related genes, but no differences remained after 24 h. At either time point, XRCT did not affect either root antioxidative enzyme activities, or the composition of rhizosphere bacterial microbiome and microbial growth parameters. The potential activities of leucine aminopeptidase and phosphomonoesterase were lower at 1 h, but did not differ from the control 24 h after exposure. A time delay of 24 h after a low X-ray exposure (0.81 Gy) was sufficient to revert any effects on the observed rhizosphere systems. Our data suggest that before implementing novel experimental designs involving XRCT, a study on its impacts on the investigated processes should be conducted.

Apple replant disease (ARD) occurs worldwide in apple orchards and nurseries and leads to a severe growth and productivity decline. Despite research on the topic, its causality remains unclear. In a split-root experiment, we grew ARD-susceptible ‘M26’ apple rootstocks in different substrate combinations (+ARD: ARD soil; -ARD: gamma-irradiated ARD soil; and Control: soil with no apple history). We investigated the microbial community composition by 16S rRNA gene amplicon sequencing (bacteria and archaea) along the soil–root continuum (bulk soil, rhizosphere and rhizoplane). Significant differences in microbial community composition and structure were found between +ARD and -ARD or +ARD and Control along the soil–root continuum, even for plants exposed simultaneously to two different substrates (-ARD/+ARD and Control/+ARD). The substrates in the respective split-root compartment defined the assembly of root-associated microbial communities, being hardly influenced by the type of substrate in the respective neighbor compartment. Root-associated representatives from Actinobacteria were the most dynamic taxa in response to the treatments, suggesting a pivotal role in ARD. Altogether, we evidenced an altered state of the microbial community in the +ARD soil, displaying altered alpha- and beta-diversity, which in turn will also impact the normal development of apple rhizosphere and rhizoplane microbiota (dysbiosis), concurring with symptom appearance.

Resilience of soils, i.e., their ability to maintain functions or recover after disturbance, is closely linked to the root-soil interface, the soil's power house. However, the limited observability of key processes at the root-soil interface has so far limited our understanding of how such resilience emerges. Here, we hypothesize that resilience emerges from self-organized spatiotemporal patterns which are the result of complex and dynamic feedbacks between physical, chemical, and biological processes occurring in the rhizosphere. We propose that the combination of modern experimental and modeling techniques, with a focus on imaging approaches, allows for understanding the complex feedbacks between plant resource acquisition, microbiome-related plant health, soil carbon sequestration, and soil structure development. A prerequisite for the identification of patterns, underlying processes, and feedback loops is that joint experimental platforms are defined and investigated in their true 2D and 3D geometry along time. This applies across different scientific disciplines from soil physics/chemistry/microbiology to plant genomics/physiology and across different scales from the nano/microscopic scale of the root soil interface, over the radial profiles around single roots, up to the root architecture and plant scale. Thus, we can move beyond isolated reductionist approaches which have dominated in rhizosphere research so far.

Growth depression of Rosa plants at sites previously used to cultivate the same or closely related species is a typical symptom of rose replant disease (RRD). Currently, limited information is available on the causes and the etiology of RRD compared to apple replant disease (ARD). Thus, this study aimed at analyzing growth characteristics, root morphology, and root metabolites, as well as microbial communities in the rhizosphere of the susceptible rootstock Rosacorymbifera 'Laxa' grown in RRD-affected soil from two sites (Heidgraben and Sangerhausen), either untreated or disinfected by gamma -irradiation. In a greenhouse bioassay, plants developed significantly more biomass in the gamma -irradiated than in the untreated soils of both sites. Several plant metabolites detected in R. corymbifera 'Laxa' roots were site- and treatment-dependent. Although aloesin was recorded in significantly higher concentrations in untreated than in gamma -irradiated soils from Heidgraben, the concentrations of phenylalanine were significantly lower in roots from untreated soil of both sites. Rhizosphere microbial communities of 8-week-old plants were studied by sequencing of 16S rRNA, ITS, and cox gene fragments amplified from total community DNA. Supported by microscopic observations, sequences affiliated to the bacterial genus Streptomyces and the fungal genus Nectria were identified as potential causal agents of RRD in the soils investigated. The relative abundance of oomycetes belonging to the genus Pythiogeton showed a negative correlation to the growth of the plants. Overall, the RRD symptoms, the effects of soil treatments on the composition of the rhizosphere microbial community revealed striking similarities to findings related to ARD.

Garden rose production involves field culture for rootstock production and cultivation of grafted plants. When roses are replanted at the same site, reduced growth, stunting and discoloration of roots are observed. This phenomenon is well-known as rose replant disease (RRD), which results in considerable economic losses. Until now, the causes of RRD are largely unknown and measures to overcome RRD are lacking. In contrast to apple, research on replant disease in roses is scarce. In this study, we have carried out a greenhouse pot experiment, using either untreated or gamma irradiated RRD soils from two sites. Slow release fertilizer was added to the soils before planting seedlings of the rootstock Rosa corymbifera ‘Laxa’. After eight weeks, shoot and root growth were recorded, roots were examined by detailed microscopic analyses and secondary metabolites were analyzed by gas chromatography-mass spectrometry. Rhizosphere samples were taken and their respective microbial communities were analyzed by amplicon sequencing of the 16S rRNA gene for bacteria and archaea as well as the ITS2 and cox2 region for fungi and oomycetes, respectively. Finally, segments of surface-disinfected roots were placed on 523 medium and outgrowing endophytic bacteria were isolated and identified. For both soils, significantly higher shoot and root biomass were observed for plants growing in irradiated compared to untreated soils. Roots were darkened, deformed and finally damaged in their outer cell layers when growing in untreated RRD soil. This corresponded to higher concentrations of two catechin derivatives and gallic acid in roots in this RRD soil compared to roots in the irradiated variants. Twenty-six endophytic bacterial isolates were obtained from roots that were affiliated to 15 different bacterial genera. Rhizosphere microbial community compositions not only differed significantly between soils of the two sites, but also between treatments (untreated versus gamma irradiated) for

Long-term agricultural fertilization strategies gradually change soil properties including the associated microbial communities. Cultivated crops recruit beneficial microbes from the surrounding soil environment via root exudates. In this study, we aimed to investigate the effects of long-term fertilization strategies across field sites on the rhizosphere prokaryotic (Bacteria and Archaea) community composition and plant performance. We conducted growth chamber experiments with lettuce (Lactuca sativa L.) cultivated in soils from two long-term field experiments, each of which compared organic versus mineral fertilization strategies. 16S rRNA gene amplicon sequencing revealed the assemblage of a rhizosphere core microbiota shared in all lettuce plants across soils, going beyond differences in community composition depending on field site and fertilization strategies. The enhanced expression of several plant genes with roles in oxidative and biotic stress signalling pathways in lettuce grown in soils with organic indicates an induced physiological status in plants. Lettuce plants grown in soils with different fertilization histories were visibly free of stress symptoms and achieved comparable biomass. This suggests a positive aboveground plant response to belowground plant-microbe interactions in the rhizosphere. Besides effects of fertilization strategy and field site, our results demonstrate the crucial role of the plant in driving rhizosphere microbiota assemblage.

The role of agriculture in the transfer of drug resistant pathogens to humans is widely debated and poorly understood. Escherichia coli is a valuable indicator organism for contamination and carriage of antimicrobial resistance (AMR) in foods. Whilst whole genome sequences for E. coli from animals and associated meats are common, sequences from produce are scarce. Produce may acquire drug resistant E. coli from animal manure fertilizers, contaminated irrigation water and wildlife, particularly birds. Whole genome sequencing was used to characterize 120 tetracycline (TET) resistant E. coli from store-bought, ready-to-eat cilantro, arugula and mixed salad from two German cities. E. coli were recovered on the day of purchase and after 7 days of refrigeration. Cilantro was far more frequently contaminated with TET-resistant E. coli providing 102 (85%) sequenced strains. Phylogroup B1 dominated the collection (n = 84, 70%) with multi-locus sequence types B1-ST6186 (n = 37, 31%), C-ST165 (n = 17, 14%), B1-ST58 (n = 14, 12%), B1-ST641 (n = 8, 7%), and C-ST88 (n = 5, 4%) frequently identified. Notably, seven strains of diverse sequence type (ST) carried genetic indicators of ColV virulence plasmid carriage. A number of previously identified and novel integrons associated with insertion elements including IS26 were also identified. Storage may affect the lineages of E. coli isolated, however further studies are needed. Our study indicates produce predominantly carry E. coli with a commensal phylogroup and a variety of AMR and virulence-associated traits. Genomic surveillance of bacteria that contaminate produce should be a matter of public health importance in order to develop a holistic understanding of the environmental dimensions of AMR.