Inside Root Research Articles

Microbes in Agriculture: Prospects and Constraints to Their Wider Adoption and Utilization in Nutrient-Poor Environments.

Microbes such as bacteria and fungi play important roles in nutrient cycling in soils, often leading to the bioavailability of metabolically important mineral elements such as nitrogen (N), phosphorus (P), iron (Fe), and zinc (Zn). Examples of microbes with beneficial traits for plant growth promotion include mycorrhizal fungi, associative diazotrophs, and the N2-fixing rhizobia belonging to the α, β and γ class of Proteobacteria. Mycorrhizal fungi generally contribute to increasing the surface area of soil-root interface for optimum nutrient uptake by plants. However, when transformed into bacteroids inside root nodules, rhizobia also convert N2 gas in air into ammonia for use by the bacteria and their host plant. Thus, nodulated legumes can meet a high proportion of their N requirements from N2 fixation. The percentage of legume N derived from atmospheric N2 fixation varies with crop species and genotype, with reported values ranging from 50-97%, 24-67%, 66-86% 27-92%, 50-92%, and 40-75% for soybean (Gycine max), groundnut (Arachis hypogea), mung bean (Vigna radiata), pigeon pea (Cajanus cajan), cowpea (Vigna unguiculata), and Kersting's groundnut (Macrotyloma geocarpum), respectively. This suggests that N2-fixing legumes require little or no N fertilizer for growth and grain yield when grown under field conditions. Even cereals and other species obtain a substantial proportion of their N nutrition from associative and endophytic N2-fixing bacteria. For example, about 12-33% of maize N requirement can be obtained from their association with Pseudomonas, Hebaspirillum, Azospirillum, and Brevundioronas, while cucumber can obtain 12.9-20.9% from its interaction with Paenebacillus beijingensis BJ-18. Exploiting the plant growth-promoting traits of soil microbes for increased crop productivity without any negative impact on the environment is the basis of green agriculture which is done through the use of biofertilizers. Either alone or in combination with other synergistic rhizobacteria, rhizobia and arbuscular mycorrhizal (AM) fungi have been widely used in agriculture, often increasing crop yields but with occasional failures due to the use of poor-quality inoculants, and wrong application techniques. This review explores the literature regarding the plant growth-promoting traits of soil microbes, and also highlights the bottle-necks in tapping this potential for sustainable agriculture.

Anthoxanthum odoratum (Poaceae) as a bioindicator of arbuscular mycorrhizal fungi abundance prior to native revegetation on agricultural soils

ABSTRACT Mycorrhizal fungi, which form symbiotic associations with plant roots, can be critical to the successful establishment of plants. Up to 90% of land plant species form associations with arbuscular mycorrhizal fungi (AMF), which develop fungal structures inside host roots. Common site preparation practices prior to ecological restoration, such as soil disturbance and herbicide application, may lessen the availability of AMF, potentially jeopardising plant establishment. However, the state of the soil microbiome can be difficult to assess directly. This study examines the potential of the widespread pasture grass Anthoxanthum odoratum (Sweet Vernal, Poaceae) as a bioassay of AMF abundance in soils. We present a rapid clearing and staining protocol for visualising AMF structures in the roots of A. odoratum. We then compare % colonisation on roots of naturally established A. odoratum plants collected from undisturbed pastures vs. sprayed and tilled pastures. Roots of A. odoratum collected from sprayed and tilled pastures showed a significantly lower average AMF colonisation at 43.8% compared with 57.6% on roots from undisturbed pastures. However, all plants sourced from the sprayed and tilled pastures still showed some AMF structures. Our results strongly suggest that A. odoratum can be a useful bioindicator of AMF presence and abundance in soils, offering a promising tool for seed-based restoration on retired agricultural land.

Phosphorus-Solubilizing Bacteria Enhance Cadmium Immobilization and Gene Expression in Wheat Roots to Reduce Cadmium Uptake.

The application of phosphorus-solubilizing bacteria is an effective method for increasing the available phosphorus content and inhibiting wheat uptake of heavy metals. However, further research is needed on the mechanism by which phosphorus-solubilizing bacteria inhibit cadmium (Cd) uptake in wheat roots and its impact on the expression of root-related genes. Here, the effects of strain Klebsiella aerogenes M2 on Cd absorption in wheat and the expression of root-related Cd detoxification and immobilization genes were determined. Compared with the control, strain M2 reduced (64.1-64.6%) Cd uptake by wheat roots. Cd fluorescence staining revealed that strain M2 blocked the entry of exogenous Cd into the root interior and enhanced the immobilization of Cd by cell walls. Forty-seven genes related to Cd detoxification, including genes encoding peroxidase, chalcone synthase, and naringenin 3-dioxygenase, were upregulated in the Cd+M2 treatment. Strain M2 enhanced the Cd resistance and detoxification activity of wheat roots through the regulation of flavonoid biosynthesis and antioxidant enzyme activity. Moreover, strain M2 regulated the expression of genes related to phenylalanine metabolism and the MAPK signaling pathway to enhance Cd immobilization in roots. These results provide a theoretical basis for the use of phosphorus-solubilizing bacteria to remediate Cd-contaminated fields and reduce Cd uptake in wheat.

Antimicrobial activity of laser assisted endodontic therapy in disinfecting root canals.

Light Amplified Stimulated Emission of Radiation (LASER) is nowadays widely studied regarding their use in endodontics and restorative dentistry. Therefore, it is of interest to evaluate the antibacterial activity of three types of LASERs namely CO2 LASER.Er, Cr:YSGG LASER and Diode LASER in disinfection of root canals. 70 patients (105 single rooted teeth) were included in the study. There was application of 2% Sodium Hypochlorite (NaOCl), 2780 nm Er,Cr:YSGG LASER, 900 nm Diode LASER and CO2 LASER. Microbial samples were collected from root canals both before and after the interventions through paper points. These parameters were evaluated in microbiology laboratory to obtain Log10 Colony Forming Units (CFUs). There was significant reduction in CFUs of microorganisms inside root canal in all three LASERs evaluated and NaOCl. The reduction in CFUs in LASERs was comparable to NaOCl. Then secondly we applied each LASER in combination with NaOCl. It was observed that reduction in CFU was greater when combination of LASER with NaOCl was applied as compared when applied alone. It can be inferred that LASER when applied with NaOCl can have significant role in disinfection of root canals.

Fast, Easy, and Comprehensive Techniques for Microscopic Observations of Fungal and Oomycete Organisms Inside the Roots of Herbaceous and Woody Plants.

The roots of herbaceous and woody plants growing in soil are complex structures that are affected by both natural and artificial fungal colonization to various extents. To obtain comprehensive information about the overall distribution of fungi or oomycetes inside a plant root system, rapid, effective, and reliable screening methods are required. To observe both fine roots, i.e., a common site for penetration of fungi and oomycetes, and mature roots, different techniques are required to overcome visual barriers, such as root browning or tissue thickening. In our protocol, we propose using fast, cost-effective, and non-harmful methods to localize fungal or oomycete structures inside plant roots. Root staining with a fluorescent dye provides a quick initial indication of the presence of fungal structures on the root surfaces. The protocol is followed by clearing and staining steps, resulting in a deeper insight into the root tissue positioning, abundance, and characteristic morphological/reproductive features of fungal or oomycete organisms. If required, the stained samples can be prepared by using freeze-drying for further observations, including advanced microscopic techniques. Key features • The protocol enhances tissue-clearing techniques employing KOH or NaOH and is applicable to a broad range of roots from different plant species. • Hydroxides are mixed with hydrogen peroxide to obtain an efficient bleaching solution, which effectively clears roots without causing significant tissue damage. • The protocol could also be used for staining of fungi or oomycetes localized both on the root surface or inside the root tissues. • Simple combination of non-fluorescent methyl blue and fluorescent solophenyl flavine dyes allows the observation of fungal organisms in both brightfield and fluorescence microscopy.

Enhancing systematic tolerance in Bermuda grass (Cynodon dactylon L.) through amplified alkB gene expression and bacterial-driven hydrocarbon degradation.

This study aimed to access the impact of soil polluted with petroleum (5, 10g petroleum kg-1 soil) on Bermuda grass (Cynodon dactylon L.) with and without applied bacterial inoculants (Arthrobacter oxydans ITRH49 and Pseudomonas sp. MixRI75). Both soil and seed were given bacterial inoculation. The evaluated morphological parameters of Bermuda grass were fresh and dry weight. The results demonstrated that applied bacterial inoculants enhanced 5.4%, 20%, 28% and 6.4%, 21%, and 29% shoot and root fresh/dry weights in Bermuda grass under controlled environment. The biochemical analysis of shoot and root was affected deleteriously by the 10g petroleum kg-1 soil pollution. Microbial inoculants enhanced the activities of enzymatic (catalase, peroxidase, glutathione reductase, ascorbate peroxidase, superoxide dismutase) and non-enzymatic (ɑ-tocopherols, proline, reduced glutathione, ascorbic acid) antioxidant to mitigate the toxic effects of ROS (H2O2) under hydrocarbon stressed condition. The maximum hydrocarbon degradation (75%) was recorded by Bermuda grass at 5g petroleum kg-1 soil contamination. Moreover, bacterial persistence and alkane hydroxylase gene (alkB) abundance and expression were observed more in the root interior than in the rhizosphere and shoot interior of Bermuda grass. Subsequently, the microbe used a biological tool to propose that the application of plant growth-promoting bacteria would be the most favorable choice in petroleum hydrocarbon polluted soil to conquer the abiotic stress in plants and the effective removal of polyaromatic hydrocarbons in polluted soil.

Arabidopsis Root Microbiome Microfluidic (ARMM) Device for Imaging Bacterial Colonization and Morphogenesis of Arabidopsis Roots.

Imaging the spatiotemporal dynamics of host-microbiota interactions is of particular interest for augmenting our understanding of these complex systems. This is especially true of plant-microbe interactions happening around, on, and inside plant roots where relatively little is understood about the dynamics of these systems. Over the past decade, a number of microfluidic devices have been developed to grow plants hydroponically in gnotobiotic conditions and image morphogenesis of the root and/or dynamics with fluorescently labeled bacteria from the plant root microbiome. Here we describe the construction and use of our Arabidopsis Root Microbiome Microfluidic (ARMM) device for imaging fluorescent protein expressing bacteria and their colonization of Arabidopsis roots. In contrast to other plant root imaging devices, we designed this device to have a larger chamber for observing Arabidopsis root elongation and plant-microbe interactions with older seedlings (between 1.5 and 4weeks after germination) and a 200μm chamber depth to specifically maintain thin Arabidopsis roots within the focal distance of the confocal microscope. Our device incorporates a new approach to growing Arabidopsis seedlings in screw-top tube caps for simplified germination and transfer to the device. We present representative images from the ARMM device including high resolution cross section images of bacterial colonization at the root surface.

Convergent gene pair dSH3 and irr regulate Pi and Fe homeostasis in Bradyrhizobium diazoefficiens USDA110 and symbiotic nitrogen fixation efficiency

The nitrogen-fixing bacteroids inhabit inside legume root nodules must manage finely the utilization of P and Fe, the two most critical elements, due to their antagonistic interactions. While the balance mechanism for them remains unclear. A double SH3 domain-containing protein (dSH3) in the Bradyrhizobium diazoefficiens USDA110 was found to inhibit the alkaline phosphatase activity, thereby reducing P supply from organophosphates. The dSH3 gene is adjacent to the irr gene, which encodes the iron response repressor and regulates Fe homeostasis under Fe-limited conditions. Their transcription directions converge to a common intergenic sequence (IGS) region, forming a convergent transcription. Extending the IGS region through Tn5 transposon or pVO155 plasmid insertion significantly down-regulated expression of this gene pair, leading to a remarkable accumulation of P and an inability to grow under Fe-limited conditions. Inoculation of soybean with either of the insertion mutants resulted in N2-fixing failure. However, the IGS-deleted mutant showed no visible changes in N2-fixing efficiency on soybean compared to that inoculated with wild type. These findings reveal a novel regulative strategy in the IGS region and its flanking convergent gene pair for antagonistic utilization of P and Fe in rhizobia and coordination of N2-fixing efficiency.

Combined biochar and wheat-derived endophytic bacteria reduces cadmium uptake in wheat grains in a metal-polluted soil

In this study, two wheat-derived cadmium (Cd)-immobilizing endophytic Pseudomonas paralactis M14 and Priestia megaterium R27 were evaluated for their effects on wheat tissue Cd uptake under hydroponic conditions. Then, the impacts of the biochar (BC), M14+R27 (MR), and BC+MR treatments on wheat Cd uptake and the mechanisms involved were investigated at the jointing, heading, and mature stages of wheat plants under field-plot conditions. A hydroponic experiment showed that the MR treatment significantly decreased the above-ground tissue Cd content compared with the M14 or R27 treatment. The BC+MR treatment reduced the grain Cd content by 51.5%–67.7% and Cd translocation factor at the mature stage of wheat plants and increased the organic matter-bound Cd content by 31%–75% in the rhizosphere soils compared with the BC or MR treatment. Compared with the BC or MR treatment, the relative abundances of the biomarkers associated with Gemmatimonas, Altererythrobacter, Gammaproteobacteria, Xanthomonadaceae, Phenylobacterium, and Nocardioides in the BC+MR-treated rhizosphere microbiome decreased and negatively correlated with the organic matter-bound Cd contents. In the BC+MR-treated root interior microbiome, the relative abundance of the biomarker belonging to Exiguobacterium increased and negatively correlated with the Cd translocation factor, while the relative abundance of the biomarker belonging to Pseudonocardiaceae decreased and positively correlated with the Cd translocation factor. Our findings suggested that the BC+MR treatment reduced Cd availability and Cd transfer through affecting the abundances of these specific biomarkers in the rhizosphere soil and root interior microbiomes, leading to decreased wheat grain Cd uptake in the contaminated soil.

Effects of Zinc Oxide Particles with Different Sizes on Root Development in Oryza sativa

Given the consistent release of zinc oxide (ZnO) nanoparticles into the environment, it is urgent to study their impact on plants in depth. In this study, grains of rice were treated with two different concentrations of ZnO nanoparticles (NP-ZnO, 10 and 100 mg/L), and their bulk counterpart (B-ZnO) were used to evaluate whether ZnO action could depend on particle size. To test this hypothesis, root growth and development assessment, oxidative stress parameters, indole-3-acetic acid (IAA) content and molecules/enzymes involved in IAA metabolism were analyzed. In situ localization of Zn in control and treated roots was also performed. Though Zn was visible inside root cells only following nanoparticle treatment, both materials (NP-ZnO and B-ZnO) were able to affect seedling growth and root morphology, with alteration in the concentration/pattern of localization of oxidative stress markers and with a different action depending on particle size. In addition, only ZnO supplied as bulk material induced a significant increase in both IAA concentration and lateral root density, supporting our hypothesis that bulk particles might enhance lateral root development through the rise of IAA concentration. Apparently, IAA concentration was influenced more by the activity of the catabolic peroxidases than by the protective action of phenols.

Effect of different intracanal medicaments on eradication of Enterococcus faecalis biofilm – Ex vivo study

Introduction: To compare the effect of different intracanal medicaments on Enterococcus faecalis (E. faecalis) biofilm inside root canals using the viable count method (colony forming unit [CFU]/mL) and scanning electron microscope (SEM). Materials and Methods: A total of 180 mandibular premolars were selected. Access cavities, cleaning and shaping were done. After sterilization, all the teeth were inoculated by E. faecalis for 5 weeks to establish a mature biofilm. The inoculated roots were equally distributed into six experimental groups corresponding to the powder used: Calcium hydroxide Ca(OH)2; metronidazole (MDZ) and chitosan. Each powder was mixed with two different vehicles, 2%chlorhexidine (CHX) solution and sterile water, as an intracanal dressing for 2 weeks. Twenty infected root canals were representing the positive control group, and other 20 noninfected root canals represented the negative control group. To determine the bacterial load (CFU/mL), samples were collected by sterile paper points pre and postdressing. Using an SEM, the root canals from each group were topographically scanned. Kruskal–Wallis and Mann–Whitney U tests (post hoc test) were used to estimate and statistically analyze the bacterial reduction data (P < 0.05). Results: Mature biofilm formation was confirmed using SEM after incubation period. MDZ + CHX and Ca(OH)2 + CHX were significantly better than other intracanal medicaments (P < 0.05). The difference in antimicrobial effectiveness between other intracanal medications was not statistically significant. Conclusion: The combination of MDZ + CHX and Ca(OH)2 + CHX recorded a significant effect against E. faecalis biofilm among the intracanal medicament used.

VRCAR-252 (INGR22088): Anthocyanin-rich petaloid-CMS line of black carrot with better heterotic potential

The storage root of cultivated carrot (Daucus carota subsp. sativus) is one of the richest sources of various pigments and has commercial significance globally. It is broadly categorized into two segments, i.e., anthocyanin/purple group (Black and Rainbow carrot) and carotenoid group (Orange, Red and Yellow carrot) on the basis of the presence of root pigments. Black carrot root is comprised with dark purple (solid purple) pigmentation of both root exterior (epidermal layer) and root interior [cortex (outer-phloem), phloem and xylem (core)]; and is one of the greatest sources of anthocyanins possessing very high antioxidative ability.

Water flow within and towards plant roots—a new concurrent solution

Abstract Various analytical models that calculate the water flow either around or inside plant roots are available, but a combined analytical solution has not yet been derived. The classical solution of Landsberg and Fowkes for water flow within a root relates the second derivative of xylem water potential to the radial water influx term. This term can be linked to well-known steady state or steady rate-based solutions for computing soil water fluxes around roots. While neglecting lateral fluxes between local depletion zones around roots, we use this link to construct a system of continuous equations that combine root internal and external water flow that can be solved numerically for two boundary conditions (specified root collar water potential and zero distal influx) and one constraint (mean bulk matric flux potential). Furthermore, an iterative matrix solution for the stepwise analytical solution of homogeneous root segments is developed. Besides accounting for soil water flow iteratively, the intrinsic effect of variable axial conductance is accounted simultaneously. The reference and the iterative matrix solution are compared for different types of corn roots, soil textures and soil dryness states, which showed good correspondence. This also revealed the importance of accounting for variable axial conductance in more detail. The proposed reference solution can be used for the evaluation of different morphological and hydraulic designs of single or multiple parallel-connected roots operating in targeted soil environments. Some details of the iterative matrix solution may be adopted in analytical–numerical solutions of water flow in complex root systems.

Bacillibactin siderophore induces iron mobilisation responses inside aerobic rice variety through YSL15 transporter

Iron uptake in Oryza sativa primarily occurs with the help of phytosiderophore i.e., deoxymugineic acid in aerobic condition. Internalisation of phytosiderophore-iron complex occurs through YSL15. However, aerobic rice at high pH faces iron deficit condition due to low secretion of plant siderophore and its early diminution. There might be a possibility that microbial siderophore of predominant rhizobacteria present in soil around rice may assist in iron uptake and transport through same transporter providing survival advantage to the plant. Present study aims to explore role of siderophore of Bacillus subtilis in improving root architecture and iron uptake in aerobic rice at high pH and explore possible transporters involved in transporting siderophore-Fe3+ complex inside rice root from rhizosphere. Identification by chrome azurol sulphonate assay and characterisation of purified extract from B. subtilis culture through LC-ESI MS confirmed presence of catecholate siderophore, Bacillibactin. Aerobic rice variety viz. Swarna Shreya grown with 5 ng/ml bacillibactin showed improved morphology and anatomy than untreated sample at alkaline pH 7.5. This is possibly due to enhanced intracellular iron content as observed in atomic absorption spectroscopy. Whole root proteomic analysis of rice root by using LC-ESI MS/MS revealed presence of YSL like transporter and siderophore import proteins in treated sample. RT-qPCR analysis showed enhanced expression of gene YSL15 in siderophore treated sample than control (pH 7.5). The present study possibly suggest role of foreign siderophore (xenosiderophore) in plant growth and development through iron uptake and transport inside rice.

Mycena species can be opportunist-generalist plant root invaders.

Traditional strict separation of fungi into ecological niches as mutualist, parasite or saprotroph is increasingly called into question. Sequences of assumed saprotrophs have been amplified from plant root interiors, and several saprotrophic genera can invade and interact with host plants in laboratory growth experiments. However, it is uncertain if root invasion by saprotrophic fungi is a widespread phenomenon and if laboratory interactions mirror field conditions. Here, we focused on the widespread and speciose saprotrophic genus Mycena and performed (1) a systematic survey of their occurrences (in ITS1/ITS2 datasets) in mycorrhizal roots of 10 plant species, and (2) an analysis of natural abundances of 13 C/15 N stable isotope signatures of Mycena basidiocarps from five field locations to examine their trophic status. We found that Mycena was the only saprotrophic genus consistently found in 9 out of 10 plant host roots, with no indication that the host roots were senescent or otherwise vulnerable. Furthermore, Mycena basidiocarps displayed isotopic signatures consistent with published 13 C/15 N profiles of both saprotrophic and mutualistic lifestyles, supporting earlier laboratory-based studies. We argue that Mycena are widespread latent invaders of healthy plant roots and that Mycena species may form a spectrum of interactions besides saprotrophy also in the field.

Root Absorption and Limited Mobility of Mandipropamid as Compared to Oxathiapiprolin and Mefenoxam After Soil Treatment of Citrus Plants for Managing Phytophthora Root Rot.

Phytophthora root rot can greatly impact citrus production worldwide, especially in newly established orchards by reducing crop yield and increasing the cost of disease management. Mandipropamid is an Oomycota fungicide that is currently registered as a soil treatment for citrus nursery container plants to manage Phytophthora root rot. In this study, we investigated the uptake of mandipropamid into citrus roots and its translocation to stems and leaves after soil application and evaluated its mobility in roots as compared to oxathiapiprolin and mefenoxam using split-root potted plants and trees in the field. A bioassay and liquid chromatography-tandem mass spectrometry were used to detect and quantify fungicides in citrus tissues, and overall, similar results were obtained using the two methods. When applied to the soil of potted, 6- to 7-month-old citrus plants using labeled rates, the majority of mandipropamid was found in root tissues (4.9 to 18.1 μg/g), but small amounts were also present in stems (0.18 to 0.32 μg/g) and leaves (0.03 to 0.22 μg/g). There was no significant increase in concentrations in all three tissues between 1 and 4 weeks after application. Concentrations in all tissues exceeded established EC50 values for mycelial growth inhibition of P. citrophthora and P. nicotianae, the main citrus root rot pathogens in California. In a split-root study where the root systems of single plants were separated, no basipetal phloem-based mobility of mandipropamid or oxathiapiprolin was observed, but relative uptake into roots was higher for mandipropamid. In contrast, low amounts of mefenoxam were also present in roots in the untreated soil. Similar results were obtained in a field study where part of the root system was treated, and fungicides were extracted from nontreated roots. All three fungicides persisted inside roots over the 8-week period of this study. Uptake and persistence inside roots, as well as the previously reported high efficacy against citrus root rot in greenhouse and field studies support the use of mandipropamid in citrus nurseries and potentially in the orchard.

Degradation of 8:2 fluorotelomer carboxylic acid (8:2 FTCA) by plants and their co-existing microorganisms

8:2 fluorotelomer carboxylic acid (8:2 FTCA), an important precursor of perfluorocarboxylic acids (PFCAs), is widely detected in environment and biotas. Hydroponic exposures were conducted to investigate the accumulation and metabolism of 8:2 FTCA in wheat (Triticum aestivum L.) and pumpkin (Cucurbita maxima L.). Endophytic and rhizospheric microorganisms co-existing with the plants were isolated to investigate their contributions to degrade 8:2 FTCA. Wheat and pumpkin roots could take up 8:2 FTCA efficiently with the root concentration factor (RCF) as 5.78 and 8.93, respectively. 8:2 FTCA could be biotransformed to 8:2 fluorotelomer unsaturated carboxylic acid (8:2 FTUCA), 7:3 fluorotelomer carboxylic acid (7:3 FTCA), and seven PFCAs with 2–8 carbon chain length in plant roots and shoots. Cytochromes P450 (CYP450) and glutathione-S-transferase (GST) activities in plants were significantly increased, while flavin-dependent monooxygenases (FMOs) activities were not changed, suggesting that CYP 450 and GST were involved in the transformation of 8:2 FTCA in plant tissues. Twelve 8:2 FTCA-degrading endophytic (8 strains) and rhizospheric (4 strains) bacterial strains were isolated from root interior, shoot interior and rhizosphere of plants, respectively. These bacteria were identified as Klebsiella sp. based on the morphology and 16S rDNA sequence, and they could biodegrade 8:2 FTCA to intermediates and stable PFCAs.

Optimization of Molecular Methods for Detecting Duckweed-Associated Bacteria.

The bacterial colonization dynamics of plants can differ between phylogenetically similar bacterial strains and in the context of complex bacterial communities. Quantitative methods that can resolve closely related bacteria within complex communities can lead to a better understanding of plant-microbe interactions. However, current methods often lack the specificity to differentiate phylogenetically similar bacterial strains. In this study, we describe molecular strategies to study duckweed-associated bacteria. We first systematically optimized a bead-beating protocol to co-isolate nucleic acids simultaneously from duckweed and bacteria. We then developed a generic fingerprinting assay to detect bacteria present in duckweed samples. To detect specific duckweed-bacterium associations, we developed a genomics-based computational pipeline to generate bacterial strain-specific primers. These strain-specific primers differentiated bacterial strains from the same genus and enabled the detection of specific duckweed-bacterium associations present in a community context. Moreover, we used these strain-specific primers to quantify the bacterial colonization of duckweed by normalization to a plant reference gene and revealed differences in colonization levels between strains from the same genus. Lastly, confocal microscopy of inoculated duckweed further supported our PCR results and showed bacterial colonization of the duckweed root-frond interface and root interior. The molecular methods introduced in this work should enable the tracking and quantification of specific plant-microbe associations within plant-microbial communities.

Tissue-specific regulation of lipid polyester synthesis genes controlling oxygen permeation into Lotus japonicus nodules

Legumes establish endosymbiotic associations with nitrogen-fixing rhizobia, which they host inside root nodules. Here, specific physiological and morphological adaptations, such as the production of oxygen-binding leghemoglobin proteins and the formation of an oxygen diffusion barrier in the nodule periphery, are essential to protect the oxygen-labile bacterial nitrogenase enzyme. The molecular basis of the latter process remains elusive as the identification of required genes is limited by the epistatic effect of nodule organogenesis over nodule infection and rhizobia accommodation. We overcame this by exploring the phenotypic diversity of Lotus japonicus accessions that uncouple nodule organogenesis from nodule infection when inoculated with a subcompatible Rhizobium strain. Using comparative transcriptomics, we identified genes with functions associated with oxygen homeostasis and deposition of lipid polyesters on cell walls to be specifically up-regulated in infected compared to noninfected nodules. As hydrophobic modification of cell walls is pivotal for creating diffusion barriers like the root endodermis, we focused on two Fatty acyl-CoA Reductase genes that were specifically activated in the root and/or in the nodule endodermis. Mutant lines in a Fatty acyl-CoA Reductase gene expressed exclusively in the nodule endodermis had decreased deposition of polyesters on this cell layer and increased nodule permeability compared to wild-type plants. Oxygen concentrations were significantly increased in the inner cortex of mutant nodules, which correlated with reduced nitrogenase activity, and impaired shoot growth. These results provide the first genetic evidence for the formation of the nodule oxygen diffusion barrier, a key adaptation enabling nitrogen fixation in legume nodules.

Identification and Functional Analysis of Tomato MicroRNAs in the Biocontrol Bacterium Pseudomonas putida Induced Plant Resistance to Meloidogyne incognita.

Root-knot nematodes (RKNs, Meloidogyne spp.) seriously damage tomato production worldwide, and biocontrol bacteria can induce tomato immunity to RKNs. Our previous studies have revealed that Pseudomonas putida strain Sneb821 can trigger tomato immunity against M. incognita and that several long noncoding RNAs and microRNAs (miRNAs) are involved in this process. However, the molecular functions of the miRNAs in tomato immune responses remain unclear. In this study, deep small RNA sequencing identified 78 differentially expressed miRNAs in tomato plants inoculated with Sneb821 and M. incognita relative to plants inoculated with M. incognita alone; 38 miRNAs were upregulated, and 40 miRNAs were downregulated. The expression levels of six known miRNAs and five novel miRNAs were validated using RT-qPCR assays. These included Sly-miR482d-3p, Sly-miR156e-5p, Sly-miR319a, novel_miR_116, novel_miR_121, and novel_miR_221, which were downregulated, and Sly-miR390a-3p, Sly-miR394-3p, Sly-miR396a-3p, novel_miR_215, and novel_miR_83, which were upregulated in plants treated with Sneb821 and M. incognita. In addition, Sly-miR482d was functionally characterized through gene silencing and overexpression of its target gene NBS-LRR (Solyc05g009750.1) in tomato and by challenging the plants with M. incognita inoculation. The number of second-stage juveniles (J2) inside roots and induced galls were significantly decreased in both Sly-miR482d-silenced plants and Solyc05g009750.1 overexpressing plants, whereas the activity of superoxide dismutase, peroxidase, and hydrogen peroxide content were significantly increased. The results suggest that Sneb821 could inhibit Sly-miR482d expression and thus regulate tomato immune responses against M. incognita infestation. This study provides novel insights into the biocontrol bacteria-mediated tomato immunity to M. incognita that engages with plant miRNAs.