Rhizosphere competent inoculants modulate the apple root-associated microbiome and plant phytoalexins.

Modulating the soil microbiome by applying microbial inoculants has gained increasing attention as eco-friendly option to improve soil disease suppressiveness. Currently, studies unraveling the interplay of inoculants, root-associated microbiome, and plant response are lacking for apple trees. Here, we provide insights into the ability of Bacillus velezensis FZB42 or Pseudomonas sp. RU47 to colonize apple root-associated microhabitats and to modulate their microbiome. We applied the two strains to apple plants grown in soils from the same site either affected by apple replant disease (ARD) or not (grass), screened their establishment by selective plating, and measured phytoalexins in roots 3, 16, and 28 days post inoculation (dpi). Sequencing of 16S rRNA gene and ITS fragments amplified from DNA extracted 28 dpi from different microhabitat samples revealed significant inoculation effects on fungal ß-diversity in root-affected soil and rhizoplane. Interestingly, only in ARD soil, most abundant bacterial amplicon sequence variants (ASVs) changed significantly in relative abundance. Relative abundances of ASVs affiliated with Enterobacteriaceae were higher in rhizoplane of apple grown in ARD soil and reduced by both inoculants. Bacterial communities in the root endosphere were not affected by the inoculants but their presence was indicated. Interestingly and previously unobserved, apple plants responded to the inoculants with increased phytoalexin content in roots, more pronounced in grass than ARD soil. Altogether, our results indicate that FZB42 and RU47 were rhizosphere competent, modulated the root-associated microbiome, and were perceived by the apple plants, which could make them interesting candidates for an eco-friendly mitigation strategy of ARD. KEY POINTS: • Rhizosphere competent inoculants modulated the microbiome (mainly fungi) • Inoculants reduced relative abundance of Enterobacteriaceae in the ARD rhizoplane • Inoculants increased phytoalexin content in roots, stronger in grass than ARD soil.

Correlative Imaging of the Rhizosphere─A Multimethod Workflow for Targeted Mapping of Chemical Gradients.

Examining in situ processes in the soil rhizosphere requires spatial information on physical and chemical properties under undisturbed conditions. We developed a correlative imaging workflow for targeted sampling of roots in their three-dimensional (3D) context and assessed the imprint of roots on chemical properties of the root-soil contact zone at micrometer to millimeter scale. Maize (Zea mays) was grown in 15N-labeled soil columns and pulse-labeled with 13CO2 to visualize the spatial distribution of carbon inputs and nitrogen uptake together with the redistribution of other elements. Soil columns were scanned by X-ray computed tomography (X-ray CT) at low resolution (45 µm) to enable image-guided subsampling of specific root segments. Resin-embedded subsamples were then analyzed by X-ray CT at high resolution (10 µm) for their 3D structure and chemical gradients around roots using micro-X-ray fluorescence spectroscopy (µXRF), nanoscale secondary ion mass spectrometry (NanoSIMS), and laser-ablation isotope ratio mass spectrometry (LA-IRMS). Concentration gradients, particularly of calcium and sulfur, with different spatial extents could be identified by µXRF. NanoSIMS and LA-IRMS detected the release of 13C into soil up to a distance of 100 µm from the root surface, whereas 15N accumulated preferentially in the root cells. We conclude that combining targeted sampling of the soil-root system and correlative microscopy opens new avenues for unraveling rhizosphere processes in situ.

Compatibility of X-ray computed tomography with plant gene expression, rhizosphere bacterial communities and enzyme activities.

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 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 the 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 reverse any effects on the observed rhizosphere systems. Our data suggest that before implementing novel experimental designs involving XRCT, a study on its impact on the investigated processes should be conducted.

Apple Replant Disease: Causes and Mitigation Strategies.

After replanting apple (Malus domestica Borkh.) on the same site severe growth suppressions, and a decline in yield and fruit quality are observed in all apple producing areas worldwide. The causes of this complex phenomenon, called apple replant disease (ARD), are only poorly understood up to now which is in part due to inconsistencies in terms and methodologies. Therefore we suggest the following definition for ARD: ARD describes a harmfully disturbed physiological and morphological reaction of apple plants to soils that faced alterations in their (micro-) biome due to the previous apple cultures. The underlying interactions likely have multiple causes that extend beyond common analytical tools in microbial ecology. They are influenced by soil properties, faunal vectors, and trophic cascades, with genotype-specific effects on plant secondary metabolism, particularly phytoalexin biosynthesis. Yet, emerging tools allow to unravel the soil and rhizosphere (micro-) biome, to characterize alterations of habitat quality, and to decipher the plant reactions. Thereby, deep insights into the reactions taking place at the root rhizosphere interface will be gained. Counteractions are suggested, taking into account that culture management should emphasize on improving soil microbial and faunal diversity as well as habitat quality rather than focus on soil disinfection.

Variable silicon accumulation in plants affects terrestrial carbon cycling by controlling lignin synthesis.

Current climate and land-use changes affect regional and global cycles of silicon (Si), with yet uncertain consequences for ecosystems. The key role of Si in marine ecology by controlling algae growth is well recognized but research on terrestrial ecosystems neglected Si since not considered an essential plant nutrient. However, grasses and various other plants accumulate large amounts of Si, and recently it has been hypothesized that incorporation of Si as a structural plant component may substitute for the energetically more expensive biosynthesis of lignin. Herein, we provide evidence supporting this hypothesis. We demonstrate that in straw of rice (Oryza sativa) deriving from a large geographic gradient across South-East Asia, the Si concentrations (ranging from 1.6% to 10.7%) are negatively related to the concentrations of carbon (31.3% to 42.5%) and lignin-derived phenols (32 to 102 mg/g carbon). Less lignin may explain results of previous studies that Si-rich straw decomposes faster. Hence, Si seems a significant but hardly recognized factor in organic carbon cycling through grasslands and other ecosystems dominated by Si-accumulating plants.

Maize (Zea mays L.) root exudation profiles change in quality and quantity during plant development - A field study.

Deciphering root exudate composition of soil-grown plants is considered a crucial step to better understand plant-soil-microbe interactions affecting plant growth performance. In this study, two genotypes of Zea mays L. (WT, rth3) differing in root hair elongation were grown in the field in two substrates (sand, loam) in custom-made, perforated columns inserted into the field plots. Root exudates were collected at different plant developmental stages (BBCH 14, 19, 59, 83) using a soil-hydroponic-hybrid exudation sampling approach. Exudates were characterized by LC-MS based non-targeted metabolomics, as well as by photometric assays targeting total dissolved organic carbon, soluble carbohydrates, proteins, amino acids, and phenolics. Results showed that plant developmental stage was the main driver shaping both the composition and quantity of exuded compounds. Carbon (C) exudation per plant increased with increasing biomass production over time, while C exudation rate per cm² root surface area h-1 decreased with plant maturity. Furthermore, exudation rates were higher in the substrate with lower nutrient mobility (i.e., loam). Surprisingly, we observed higher exudation rates in the root hairless rth3 mutant compared to the root hair-forming WT sibling, though exudate metabolite composition remained similar. Our results highlight the impact of plant developmental stage on the plant-soil-microbe interplay.

Plasticity of rhizosphere hydraulic properties as a key for efficient utilization of scarce resources.

BACKGROUND: It is known that the soil near roots, the so-called rhizosphere, has physical and chemical properties different from those of the bulk soil. Rhizosphere properties are the result of several processes: root and soil shrinking/swelling during drying/wetting cycles, soil compaction by root growth, mucilage exuded by root caps, interaction of mucilage with soil particles, mucilage shrinking/swelling and mucilage biodegradation. These processes may lead to variable rhizosphere properties, i.e. the presence of air-filled gaps between soil and roots; water repellence in the rhizosphere caused by drying of mucilage around the soil particles; or water accumulation in the rhizosphere due to the high water-holding capacity of mucilage. The resulting properties are not constant in time but they change as a function of soil condition, root growth rate and mucilage age. SCOPE: We consider such a variability as an expression of rhizosphere plasticity, which may be a strategy for plants to control which part of the root system will have a facilitated access to water and which roots will be disconnected from the soil, for instance by air-filled gaps or by rhizosphere hydrophobicity. To describe such a dualism, we suggest classifying rhizosphere into two categories: class A refers to a rhizosphere covered with hydrated mucilage that optimally connects roots to soil and facilitates water uptake from dry soils. Class B refers to the case of air-filled gaps and/or hydrophobic rhizosphere, which isolate roots from the soil and may limit water uptake from the soil as well water loss to the soil. The main function of roots covered by class B will be long-distance transport of water. OUTLOOK: This concept has implications for soil and plant water relations at the plant scale. Root water uptake in dry conditions is expected to shift to regions covered with rhizosphere class A. On the other hand, hydraulic lift may be limited in regions covered with rhizosphere class B. New experimental methods need to be developed and applied to different plant species and soil types, in order to understand whether such dualism in rhizosphere properties is an important mechanism for efficient utilization of scarce resources and drought tolerance.

Plant Age and Soil Texture Rather Than the Presence of Root Hairs Cause Differences in Maize Resource Allocation and Root Gene Expression in the Field.

Understanding the biological roles of root hairs is key to projecting their contributions to plant growth and to assess their relevance for plant breeding. The objective of this study was to assess the importance of root hairs for maize nutrition, carbon allocation and root gene expression in a field experiment. Applying wild type and root hairless rth3 maize grown on loam and sand, we examined the period of growth including 4-leaf, 9-leaf and tassel emergence stages, accompanied with a low precipitation rate. rth3 maize had lower shoot growth and lower total amounts of mineral nutrients than wild type, but the concentrations of mineral elements, root gene expression, or carbon allocation were largely unchanged. For these parameters, growth stage accounted for the main differences, followed by substrate. Substrate-related changes were pronounced during tassel emergence, where the concentrations of several elements in leaves as well as cell wall formation-related root gene expression and C allocation decreased. In conclusion, the presence of root hairs stimulated maize shoot growth and total nutrient uptake, but other parameters were more impacted by growth stage and soil texture. Further research should relate root hair functioning to the observed losses in maize productivity and growth efficiency.

Three-dimensional visualization and quantification of water content in the rhizosphere.

• Despite the importance of rhizosphere properties for water flow from soil to roots, there is limited quantitative information on the distribution of water in the rhizosphere of plants. • Here, we used neutron tomography to quantify and visualize the water content in the rhizosphere of the plant species chickpea (Cicer arietinum), white lupin (Lupinus albus), and maize (Zea mays) 12 d after planting. • We clearly observed increasing soil water contents (θ) towards the root surface for all three plant species, as opposed to the usual assumption of decreasing water content. This was true for tap roots and lateral roots of both upper and lower parts of the root system. Furthermore, water gradients around the lower part of the roots were smaller and extended further into bulk soil compared with the upper part, where the gradients in water content were steeper. • Incorporating the hydraulic conductivity and water retention parameters of the rhizosphere into our model, we could simulate the gradual changes of θ towards the root surface, in agreement with the observations. The modelling result suggests that roots in their rhizosphere may modify the hydraulic properties of soil in a way that improves uptake under dry conditions.

An improved method for the segmentation of roots from X-ray computed tomography 3D images: Rootine v.2.

BACKGROUND: X-ray computed tomography is acknowledged as a powerful tool for the study of root system architecture of plants growing in soil. In this paper, we improved the original root segmentation algorithm "Rootine" and present its succeeding version "Rootine v.2". In addition to gray value information, Rootine algorithms are based on shape detection of cylindrical roots. Both algorithms are macros for the ImageJ software and are made freely available to the public. New features in Rootine v.2 are (i) a pot wall detection and removal step to avoid segmentation artefacts for roots growing along the pot wall, (ii) a calculation of the root average gray value based on a histogram analysis, (iii) an automatic calculation of thresholds for hysteresis thresholding of the tubeness image to reduce the number of parameters and (iv) a false negatives recovery based on shape criteria to increase root recovery. We compare the segmentation results of Rootine v.1 and Rootine v.2 with the results of root washing and subsequent analysis with WinRhizo. We use a benchmark dataset of maize roots (Zea mays L. cv. B73) grown in repacked soil for two scenarios with differing soil heterogeneity and image quality. RESULTS: We demonstrate that Rootine v.2 outperforms its preceding version in terms of root recovery and enables to match better the root diameter distribution data obtained with root washing. Despite a longer processing time, Rootine v.2 comprises less user-defined parameters and shows an overall greater usability. CONCLUSION: The proposed method facilitates higher root detection accuracy than its predecessor and has the potential for improving high-throughput root phenotyping procedures based on X-ray computed tomography data analysis.

Direct Imaging of Plant Metabolites in the Rhizosphere Using Laser Desorption Ionization Ultra-High Resolution Mass Spectrometry.

The interplay of rhizosphere components such as root exudates, microbes, and minerals results in small-scale gradients of organic molecules in the soil around roots. The current methods for the direct chemical imaging of plant metabolites in the rhizosphere often lack molecular information or require labeling with fluorescent tags or isotopes. Here, we present a novel workflow using laser desorption ionization (LDI) combined with mass spectrometric imaging (MSI) to directly analyze plant metabolites in a complex soil matrix. Undisturbed samples of the roots and the surrounding soil of Zea mays L. plants from either field- or laboratory-scale experiments were embedded and cryosectioned to 100 µm thin sections. The target metabolites were detected with a spatial resolution of 25 µm in the root and the surrounding soil based on accurate masses using ultra-high mass resolution laser desorption ionization Fourier-transform ion cyclotron resonance mass spectrometry (LDI-FT-ICR-MS). Using this workflow, we could determine the rhizosphere gradients of a dihexose (e.g., sucrose) and other plant metabolites (e.g., coumaric acid, vanillic acid). The molecular gradients for the dihexose showed a high abundance of this metabolite in the root and a strong depletion of the signal intensity within 150 µm from the root surface. Analyzing several sections from the same undisturbed soil sample allowed us to follow molecular gradients along the root axis. Benefiting from the ultra-high mass resolution, isotopologues of the dihexose could be readily resolved to enable the detection of stable isotope labels on the compound level. Overall, the direct molecular imaging via LDI-FT-ICR-MS allows for the first time a non-targeted or targeted analysis of plant metabolites in undisturbed soil samples, paving the way to study the turnover of root-derived organic carbon in the rhizosphere with high chemical and spatial resolution.

Spatiotemporal Dynamics of Maize (Zea mays L.) Root Growth and Its Potential Consequences for the Assembly of the Rhizosphere Microbiota.

Numerous studies have shown that plants selectively recruit microbes from the soil to establish a complex, yet stable and quite predictable microbial community on their roots - their "microbiome." Microbiome assembly is considered as a key process in the self-organization of root systems. A fundamental question for understanding plant-microbe relationships is where a predictable microbiome is formed along the root axis and through which microbial dynamics the stable formation of a microbiome is challenged. Using maize as a model species for which numerous data on dynamic root traits are available, this mini-review aims to give an integrative overview on the dynamic nature of root growth and its consequences for microbiome assembly based on theoretical considerations from microbial community ecology.

Soil Texture, Sampling Depth and Root Hairs Shape the Structure of ACC Deaminase Bacterial Community Composition in Maize Rhizosphere.

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.

Assembly Patterns of the Rhizosphere Microbiome Along the Longitudinal Root Axis of Maize (Zea mays L.).

It is by now well proven that different plant species within their specific root systems select for distinct subsets of microbiota from bulk soil - their individual rhizosphere microbiomes. In maize, root growth advances several centimeters each day, with the locations, quality and quantity of rhizodeposition changing. We investigated the assembly of communities of prokaryotes (archaea and bacteria) and their protistan predators (Cercozoa, Rhizaria) along the longitudinal root axis of maize (Zea mays L.). We grew maize plants in an agricultural loamy soil and sampled rhizosphere soil at distinct locations along maize roots. We applied high-throughput sequencing, followed by diversity and network analyses in order to track changes in relative abundances, diversity and co-occurrence of rhizosphere microbiota along the root axis. Apart from a reduction of operational taxonomic unit (OTU) richness and a strong shift in community composition between bulk soil and root tips, patterns of microbial community assembly along maize-roots were more complex than expected. High variation in beta diversity at root tips and the root hair zone indicated substantial randomness of community assembly. Root hair zone communities were characterized by massive co-occurrence of microbial taxa, likely fueled by abundant resource supply from rhizodeposition. Further up the root where lateral roots emerged processes of community assembly appeared to be more deterministic (e.g., through competition and predation). This shift toward significance of deterministic processes was revealed by low variability of beta diversity, changes in network topology, and the appearance of regular phylogenetic co-occurrence patterns in bipartite networks between prokaryotes and their potential protistan predators. Such patterns were strongest in regions with fully developed laterals, suggesting that a consistent rhizosphere microbiome finally assembled. For the targeted improvement of microbiome function, such knowledge on the processes of microbiome assembly on roots and its temporal and spatial variability is crucially important.

Root exposure to apple replant disease soil triggers local defense response and rhizoplane microbiome dysbiosis.

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.

Dynamics of localised nitrogen supply and relevance for root growth of Vicia faba ('Fuego') and Hordeum vulgare ('Marthe') in soil.

Root growth responds to local differences in N-form and concentration. This is known for artificial systems and assumed to be valid in soil. The purpose of this study is to challenge this assumption for soil mesocosms locally supplied with urea with and without nitrification inhibitor. Soil column experiments with Vicia faba ('Fuego') and Hordeum vulgare ('Marthe') were performed to investigate soil solution chemistry and root growth response of these two species with contrasting root architectures to the different N-supply simultaneously. Root growth was analysed over time and separately for the fertiliser layer and the areas above and below with X-ray CT (via region growing) and WinRHIZO. Additionally, NO3- and NH4+ in soil and soil solution were analysed. In Vicia faba, no pronounced differences were observed, although CT analysis indicated different root soil exploration for high NH4+. In Hordeum vulgare, high NO3- inhibited lateral root growth while high NH4+ stimulated the formation of first order laterals. The growth response to locally distributed N-forms in soil is species specific and less pronounced than in artificial systems. The combination of soil solution studies and non-invasive imaging of root growth can substantially improve the mechanistic understanding of root responses to different N-forms in soil.