Synergistic effects of plant genotype and soil microbiome on growth in Lotus japonicus.

The biological interactions between plants and their root microbiomes are essential for plant growth, and even though plant genotype (G), soil microbiome (M), and growth conditions (environment; E) are the core factors shaping root microbiome, their relationships remain unclear. In this study, we investigated the effects of G, M, and E and their interactions on the Lotus root microbiome and plant growth using an in vitro cross-inoculation approach, which reconstructed the interactions between nine Lotus accessions and four soil microbiomes under two different environmental conditions. Results suggested that a large proportion of the root microbiome composition is determined by M and E, while G-related (G, G × M, and G × E) effects were significant but small. In contrast, the interaction between G and M had a more pronounced effect on plant shoot growth than M alone. Our findings also indicated that most microbiome variations controlled by M have little effect on plant phenotypes, whereas G × M interactions have more significant effects. Plant genotype-dependent interactions with soil microbes warrant more attention to optimize crop yield and resilience.

Nitrogen and Nod factor signaling determine Lotus japonicus root exudate composition and bacterial assembly.

Symbiosis with soil-dwelling bacteria that fix atmospheric nitrogen allows legume plants to grow in nitrogen-depleted soil. Symbiosis impacts the assembly of root microbiota, but it is unknown how the interaction between the legume host and rhizobia impacts the remaining microbiota and whether it depends on nitrogen nutrition. Here, we use plant and bacterial mutants to address the role of Nod factor signaling on Lotus japonicus root microbiota assembly. We find that Nod factors are produced by symbionts to activate Nod factor signaling in the host and that this modulates the root exudate profile and the assembly of a symbiotic root microbiota. Lotus plants with different symbiotic abilities, grown in unfertilized or nitrate-supplemented soils, display three nitrogen-dependent nutritional states: starved, symbiotic, or inorganic. We find that root and rhizosphere microbiomes associated with these states differ in composition and connectivity, demonstrating that symbiosis and inorganic nitrogen impact the legume root microbiota differently. Finally, we demonstrate that selected bacterial genera characterizing state-dependent microbiomes have a high level of accurate prediction.

RinRK1 enhances NF receptors accumulation in nanodomain-like structures at root-hair tip.

Legume-rhizobia root-nodule symbioses involve the recognition of rhizobial Nod factor (NF) signals by NF receptors, triggering both nodule organogenesis and rhizobial infection. RinRK1 is induced by NF signaling and is essential for infection thread (IT) formation in Lotus japonicus. However, the precise mechanism underlying this process remains unknown. Here, we show that RinRK1 interacts with the extracellular domains of NF receptors (NFR1 and NFR5) to promote their accumulation at root hair tips in response to rhizobia or NFs. Furthermore, Flotillin 1 (Flot1), a nanodomain-organizing protein, associates with the kinase domains of NFR1, NFR5 and RinRK1. RinRK1 promotes the interactions between Flot1 and NF receptors and both RinRK1 and Flot1 are necessary for the accumulation of NF receptors at root hair tips upon NF stimulation. Our study shows that RinRK1 and Flot1 play a crucial role in NF receptor complex assembly within localized plasma membrane signaling centers to promote symbiotic infection.

A pathogenesis-related protein, PRP1, negatively regulates root nodule symbiosis in Lotus japonicus.

The legume-rhizobium symbiosis represents a unique model within the realm of plant-microbe interactions. Unlike typical cases of pathogenic invasion, the infection of rhizobia and their residence within symbiotic cells do not elicit a noticeable immune response in plants. Nevertheless, there is still much to uncover regarding the mechanisms through which plant immunity influences rhizobial symbiosis. In this study, we identify an important player in this intricate interplay: Lotus japonicus PRP1, which serves as a positive regulator of plant immunity but also exhibits the capacity to decrease rhizobial colonization and nitrogen fixation within nodules. The PRP1 gene encodes an uncharacterized protein and is named Pathogenesis-Related Protein1, owing to its orthologue in Arabidopsis thaliana, a pathogenesis-related family protein (At1g78780). The PRP1 gene displays high expression levels in nodules compared to other tissues. We observed an increase in rhizobium infection in the L. japonicus prp1 mutants, whereas PRP1-overexpressing plants exhibited a reduction in rhizobium infection compared to control plants. Intriguingly, L. japonicus prp1 mutants produced nodules with a pinker colour compared to wild-type controls, accompanied by elevated levels of leghaemoglobin and an increased proportion of infected cells within the prp1 nodules. The transcription factor Nodule Inception (NIN) can directly bind to the PRP1 promoter, activating PRP1 gene expression. Furthermore, we found that PRP1 is a positive mediator of innate immunity in plants. In summary, our study provides clear evidence of the intricate relationship between plant immunity and symbiosis. PRP1, acting as a positive regulator of plant immunity, simultaneously exerts suppressive effects on rhizobial infection and colonization within nodules.

A Novel Rhizobium sp. Chiba-1 Strain Exhibits a Host Range for Nodule Symbiosis in Lotus Species.

Rhizobia are soil bacteria that induce the formation of nodules in the roots of leguminous plants for mutualistic establishment. Although the symbiotic mechanism between Lotus japonicus and its major symbiotic rhizobia, Mesorhizobium loti, has been extensively characterized, our understanding of symbiotic mechanisms, such as host specificity and host ranges, remains limited. In the present study, we isolated a novel Rhizobium strain capable of forming nodules on L. burttii from agricultural soil at Iwate prefecture in Japan. We conducted genomic and host range ana-lyses of various Lotus species. The results obtained revealed that the novel isolated Rhizobium sp. Chiba-1 was closely related to R. leguminosarum and had a wide host range that induced nodule development, including L. burttii and several L. japonicus wild-type accessions. However, L. japonicus Gifu exhibited an incompatible nodule phenotype. We also identified the formation of an epidermal infection threads that was dependent on the Lotus species and independent of nodule organ development. In conclusion, this newly isolated Rhizobium strain displays a distinct nodulation phenotype from Lotus species, and the results obtained herein provide novel insights into the functional mechanisms underlying host specificity and host ranges.

A micro RNA mediates shoot control of root branching.

Plants extract mineral nutrients from the soil, or from interactions with mutualistic soil microbes via their root systems. Adapting root architecture to nutrient availability enables efficient resource utilization, particularly in patchy and dynamic environments. Root growth responses to soil nitrogen levels are shoot-mediated, but the identity of shoot-derived mobile signals regulating root growth responses has remained enigmatic. Here we show that a shoot-derived micro RNA, miR2111, systemically steers lateral root initiation and nitrogen responsiveness through its root target TML (TOO MUCH LOVE) in the legume Lotus japonicus, where miR2111 and TML were previously shown to regulate symbiotic infections with nitrogen fixing bacteria. Intriguingly, systemic control of lateral root initiation by miR2111 and TML/HOLT (HOMOLOGUE OF LEGUME TML) was conserved in the nonsymbiotic ruderal Arabidopsis thaliana, which follows a distinct ecological strategy. Thus, the miR2111-TML/HOLT regulon emerges as an essential, conserved factor in adaptive shoot control of root architecture in dicots.

Both incompatible and compatible rhizobia inhabit the intercellular spaces of leguminous root nodules.

In addition to rhizobia, many types of co-existent bacteria are found in leguminous root nodules, but their habitats are unclear. To investigate this phenomenon, we labeled Bradyrhizobium diazoefficiens USDA122 and Bradyrhizobium sp. SSBR45 with Discosoma sp. red fluorescent protein (DsRed) or enhanced green fluorescent protein (eGFP). USDA122 enhances soybean growth by forming effective root nodules, but SSBR45 does not form any nodules. Using low-magnification laser scanning confocal microscopy, we found that infected cells in the central zone of soybean nodules appeared to be occupied by USDA122. Notably, high-magnification microscopy after co-inoculation of non-fluorescent USDA122 and fluorescence-labeled SSBR45 also revealed that SSBR45 inhabits the intercellular spaces of healthy nodules. More unexpectedly, co-inoculation of eGFP-labeled USDA122 and DsRed-labeled SSBR45 (and vice versa) revealed the presence of USDA122 bacteria in both the symbiosomes of infected cells and in the apoplasts of healthy nodules. We then next inspected nodules formed after a mixed inoculation of differently-labeled USDA122, without SSBR45, and confirmed the inhabitation of the both populations of USDA122 in the intercellular spaces. In contrast, infected cells were occupied by single-labeled USDA122. We also observed Mesorhizobium loti in the intercellular spaces of active wild-type nodules of Lotus japonicus using transmission electron microscopy. Compatible intercellular rhizobia have been described during nodule formation of several legume species and in some mutants, but our evidence suggests that this type of colonization may occur much more commonly in leguminous root nodules.

Aromatic amino acid biosynthesis impacts root hair development and symbiotic associations in Lotus japonicus.

Legume roots can be symbiotically colonized by arbuscular mycorrhizal (AM) fungi and nitrogen-fixing bacteria. In Lotus japonicus, the latter occurs intracellularly by the cognate rhizobial partner Mesorhizobium loti or intercellularly with the Agrobacterium pusense strain IRBG74. Although these symbiotic programs show distinctive cellular and transcriptome signatures, some molecular components are shared. In this study, we demonstrate that 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase 1 (DAHPS1), the first enzyme in the biosynthetic pathway of aromatic amino acids (AAAs), plays a critical role in root hair development and for AM and rhizobial symbioses in Lotus. Two homozygous DAHPS1 mutants (dahps1-1 and dahps1-2) showed drastic alterations in root hair morphology, associated with alterations in cell wall dynamics and a progressive disruption of the actin cytoskeleton. The altered root hair structure was prevented by pharmacological and genetic complementation. dahps1-1 and dahps1-2 showed significant reductions in rhizobial infection (intracellular and intercellular) and nodule organogenesis and a delay in AM colonization. RNAseq analysis of dahps1-2 roots suggested that these phenotypes are associated with downregulation of several cell wall-related genes, and with an attenuated signaling response. Interestingly, the dahps1 mutants showed no detectable pleiotropic effects, suggesting a more selective recruitment of this gene in certain biological processes. This work provides robust evidence linking AAA metabolism to root hair development and successful symbiotic associations.

A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus.

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro, the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined ß-1,3/ß-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus. The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

Population genomics of Australian indigenous Mesorhizobium reveals diverse nonsymbiotic genospecies capable of nitrogen-fixing symbioses following horizontal gene transfer.

Mesorhizobia are soil bacteria that establish nitrogen-fixing symbioses with various legumes. Novel symbiotic mesorhizobia frequently evolve following horizontal transfer of symbiosis-gene-carrying integrative and conjugative elements (ICESyms) to indigenous mesorhizobia in soils. Evolved symbionts exhibit a wide range in symbiotic effectiveness, with some fixing nitrogen poorly or not at all. Little is known about the genetic diversity and symbiotic potential of indigenous soil mesorhizobia prior to ICESym acquisition. Here we sequenced genomes of 144 Mesorhizobium spp. strains cultured directly from cultivated and uncultivated Australian soils. Of these, 126 lacked symbiosis genes. The only isolated symbiotic strains were either exotic strains used previously as legume inoculants, or indigenous mesorhizobia that had acquired exotic ICESyms. No native symbiotic strains were identified. Indigenous nonsymbiotic strains formed 22 genospecies with phylogenomic diversity overlapping the diversity of internationally isolated symbiotic Mesorhizobium spp. The genomes of indigenous mesorhizobia exhibited no evidence of prior involvement in nitrogen-fixing symbiosis, yet their core genomes were similar to symbiotic strains and they generally lacked genes for synthesis of biotin, nicotinate and thiamine. Genomes of nonsymbiotic mesorhizobia harboured similar mobile elements to those of symbiotic mesorhizobia, including ICESym-like elements carrying aforementioned vitamin-synthesis genes but lacking symbiosis genes. Diverse indigenous isolates receiving ICESyms through horizontal gene transfer formed effective symbioses with Lotus and Biserrula legumes, indicating most nonsymbiotic mesorhizobia have an innate capacity for nitrogen-fixing symbiosis following ICESym acquisition. Non-fixing ICESym-harbouring strains were isolated sporadically within species alongside effective symbionts, indicating chromosomal lineage does not predict symbiotic potential. Our observations suggest previously observed genomic diversity amongst symbiotic Mesorhizobium spp. represents a fraction of the extant diversity of nonsymbiotic strains. The overlapping phylogeny of symbiotic and nonsymbiotic clades suggests major clades of Mesorhizobium diverged prior to introduction of symbiosis genes and therefore chromosomal genes involved in symbiosis have evolved largely independent of nitrogen-fixing symbiosis.

Structure of the lipopolysaccharide O-antigen of Serratia spp. strains 10.1WK and 1XS plant endophytes isolated from O. biennis and L. corniculatus.

O-specific polysaccharides (O-PSs) isolated from lipopolysaccharides of Serratia spp., strains 10.1WK and 1XS, which are endophytic bacteria of Oenothera biennis (common evening-primrose) and Lotus corniculatus (bird's-foot trefoil), plants growing on a petroleum hydrocarbon polluted site in the Silesia region, were investigated. The high-molecular-weight O-PS fractions liberated from lipopolysaccharides by mild acid hydrolysis were studied using chemical methods, MALDI-TOF mass spectrometry, and a set of 1D and 2D NMR spectroscopy techniques. It was found that both O-specific polysaccharides were built of an identical trisaccharide repeating unit composed of d-Rhap and d-Manp residues. The following structure of the O-PSs of Serratia spp. strains 10.1WK and 1XS was established: →4)-α-d-Rhap-(1 â†’ 3)-ß-d-Manp-(1 â†’ 4)-ß-d-Rhap-(1→.

Interplay between rhizobial nodulation and arbuscular mycorrhizal fungal colonization in Lotus japonicus roots.

AIMS: This study aims to identify main factors that influence the tripartite association of legumes with arbuscular mycorrhiza fungi (AMF) and nitrogen-fixing rhizobia. METHODS AND RESULTS: Concurrent inoculations with Mesorhizobium loti and four AMF strains were performed on the model legume Lotus japonicus. Nodulation was significantly enhanced by all AMF strains, under normal conditions, and by specific AMF strains under heat-stress conditions. The impact of rhizobia on mycorrhizal colonization was AMF strain dependent. Co-inoculation trials, where either AMF or rhizobia were restricted outside the root, showed that the symbiotic phenotypes are not influenced by microbial interactions at the pre-symbiotic stage. External application of nutrients showed that P enhances nodulation, while N application does not enhance mycorrhizal colonization. CONCLUSIONS: Nodulation and mycorhization affect one another during advanced stages of the symbiosis. AMF strains may enhance nodulation under both normal and high environmental temperatures. Rhizobium-AMF compatibility is critical, as rhizobium may positively affect specific AMF strains, an effect that does not derive from increased N uptake.

A Promiscuity Locus Confers Lotus burttii Nodulation with Rhizobia from Five Different Genera.

Legumes acquire access to atmospheric nitrogen through nitrogen fixation by rhizobia in root nodules. Rhizobia are soil-dwelling bacteria and there is a tremendous diversity of rhizobial species in different habitats. From the legume perspective, host range is a compromise between the ability to colonize new habitats, in which the preferred symbiotic partner may be absent, and guarding against infection by suboptimal nitrogen fixers. Here, we investigate natural variation in rhizobial host range across Lotus species. We find that Lotus burttii is considerably more promiscuous than Lotus japonicus, represented by the Gifu accession, in its interactions with rhizobia. This promiscuity allows Lotus burttii to form nodules with Mesorhizobium, Rhizobium, Sinorhizobium, Bradyrhizobium, and Allorhizobium species that represent five distinct genera. Using recombinant inbred lines, we have mapped the Gifu/burttii promiscuity quantitative trait loci (QTL) to the same genetic locus regardless of rhizobial genus, suggesting a general genetic mechanism for symbiont-range expansion. The Gifu/burttii QTL now provides an opportunity for genetic and mechanistic understanding of promiscuous legume-rhizobia interactions. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Effect of vacuum impregnation assisted probiotics fermentation suspension on shelf life quality of freshly cut lotus root.

Probiotic fermentation suspension was used to extend the shelf life of freshly cut lotus root for the first time, which played a dual role of biological protection and quality maintenance. Fermentation suspension contained lactic acid bacteria (8-9 log CFU/mL) was prepared from juice of lotus root and used to immerse samples under atmospheric pressure and vacuum. Probiotic fermentation suspension inhibited microorganism and the activity of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase (PAL), which slowed down the physiological reaction and was beneficial to maintain the color and hardness of tissues. Lactic acid bacteria antagonized other microorganisms, and metabolic acid production played a continuous role in preservation during storage. The vacuum was helpful for the fermentation suspension to be fully impregnated into samples. The probiotic fermentation suspension had a significant inhibitory effect on E.coli O157:H7, and extended lotus root shelf life from 3 to 9 days.

Microbiome profiling reveals that Pseudomonas antagonises parasitic nodule colonisation of cheater rhizobia in Lotus.

Nodule microbiota are dominated by symbiotic nitrogen-fixing rhizobia, however, other non-rhizobial bacteria also colonise this niche. Although many of these bacteria harbour plant-growth-promoting functions, it is not clear whether these less abundant nodule colonisers impact root-nodule symbiosis. We assessed the relationship between the nodule microbiome and nodulation as influenced by the soil microbiome, by using a metabarcoding approach to characterise the communities inside nodules of healthy and starved Lotus species. A machine learning algorithm and network analyses were used to identify nodule bacteria of interest, which were re-inoculated onto plants in controlled conditions to observe their potential functionality. The nodule microbiome of all tested species differed according to inoculum, but only that of Lotus burttii varied with plant health. Amplicon sequence variants representative of Pseudomonas species were the most indicative non-rhizobial signatures inside healthy L. burttii nodules and negatively correlated with Rhizobium sequences. A representative Pseudomonas isolate co-colonised nodules infected with a beneficial Mesorhizobium, but not with an ineffective Rhizobium isolate and another even reduced the number of ineffective nodules induced on Lotus japonicus. Our results show that nodule endophytes influence the overall outcome of the root-nodule symbiosis, albeit in a plant host-specific manner.

Acidovorax pan-genome reveals specific functional traits for plant beneficial and pathogenic plant-associations.

Beta-proteobacteria belonging to the genus Acidovorax have been described from various environments. Many strains can interact with a range of hosts, including humans and plants, forming neutral, beneficial or detrimental associations. In the frame of this study, we investigated the genomic properties of 52 bacterial strains of the genus Acidovorax, isolated from healthy roots of Lotus japonicus, with the intent of identifying traits important for effective plant-growth promotion. Based on single-strain inoculation bioassays with L. japonicus, performed in a gnotobiotic system, we distinguished seven robust plant-growth promoting strains from strains with no significant effects on plant-growth. We showed that the genomes of the two groups differed prominently in protein families linked to sensing and transport of organic acids, production of phytohormones, as well as resistance and production of compounds with antimicrobial properties. In a second step, we compared the genomes of the tested isolates with those of plant pathogens and free-living strains of the genus Acidovorax sourced from public repositories. Our pan-genomics comparison revealed features correlated with commensal and pathogenic lifestyle. We showed that commensals and pathogens differ mostly in their ability to use plant-derived lipids and in the type of secretion-systems being present. Most free-living Acidovorax strains did not harbour any secretion-systems. Overall, our data indicate that Acidovorax strains undergo extensive adaptations to their particular lifestyle by horizontal uptake of novel genetic information and loss of unnecessary genes.

Symbiotic responses of Lotus japonicus to two isogenic lines of a mycorrhizal fungus differing in the presence/absence of an endobacterium.

As other arbuscular mycorrhizal fungi, Gigaspora margarita contains unculturable endobacteria in its cytoplasm. A cured fungal line has been obtained and showed it was capable of establishing a successful mycorrhizal colonization. However, previous OMICs and physiological analyses have demonstrated that the cured fungus is impaired in some functions during the pre-symbiotic phase, leading to a lower respiration activity, lower ATP, and antioxidant production. Here, by combining deep dual-mRNA sequencing and proteomics applied to Lotus japonicus roots colonized by the fungal line with bacteria (B+) and by the cured line (B-), we tested the hypothesis that L. japonicus (i) activates its symbiotic pathways irrespective of the presence or absence of the endobacterium, but (ii) perceives the two fungal lines as different physiological entities. Morphological observations confirmed the absence of clear endobacteria-dependent changes in the mycorrhizal phenotype of L. japonicus, while transcript and proteomic datasets revealed activation of the most important symbiotic pathways. They included the iconic nutrient transport and some less-investigated pathways, such as phenylpropanoid biosynthesis. However, significant differences between the mycorrhizal B+/B- plants emerged in the respiratory pathways and lipid biosynthesis. In both cases, the roots colonized by the cured line revealed a reduced capacity to activate genes involved in antioxidant metabolism, as well as the early biosynthetic steps of the symbiotic lipids, which are directed towards the fungus. Similar to its pre-symbiotic phase, the intraradical fungus revealed transcripts related to mitochondrial activity, which were downregulated in the cured line, as well as perturbation in lipid biosynthesis.

Nitric Oxide Detoxification by Mesorhizobium loti Affects Root Nodule Symbiosis with Lotus japonicus.

Root nodule symbiosis between legumes and rhizobia involves nitric oxide (NO) regulation by both the host plant and symbiotic rhizobia. However, the mechanisms by which the rhizobial control of NO affects root nodule symbiosis in Lotus japonicus are unknown. Therefore, we herein investigated the effects of enhanced NO removal by Mesorhizobium loti on symbiosis with L. japonicus. The hmp gene, which in Sinorhizobium meliloti encodes a flavohemoglobin involved in NO detoxification, was introduced into M. loti to generate a transconjugant with enhanced NO removal. The symbiotic phenotype of the transconjugant with L. japonicus was examined. The transconjugant showed delayed infection and higher nitrogenase activity in mature nodules than the wild type, whereas nodule senescence was normal. This result is in contrast to previous findings showing that enhanced NO removal in L. japonicus by class 1 phytoglobin affected nodule senescence. To evaluate differences in NO detoxification between M. loti and L. japonicus, NO localization in nodules was investigated. The enhanced expression of class 1 phytoglobin in L. japonicus reduced the amount of NO not only in infected cells, but also in vascular bundles, whereas that of hmp in M. loti reduced the amount of NO in infected cells only. This difference suggests that NO detoxification by M. loti exerts different effects in symbiosis than that by L. japonicus.

Host preference and invasiveness of commensal bacteria in the Lotus and Arabidopsis root microbiota.

Roots of different plant species are colonized by bacterial communities, that are distinct even when hosts share the same habitat. It remains unclear to what extent the host actively selects these communities and whether commensals are adapted to a specific plant species. To address this question, we assembled a sequence-indexed bacterial culture collection from roots and nodules of Lotus japonicus that contains representatives of most species previously identified using metagenomics. We analysed taxonomically paired synthetic communities from L. japonicus and Arabidopsis thaliana in a multi-species gnotobiotic system and detected signatures of host preference among commensal bacteria in a community context, but not in mono-associations. Sequential inoculation experiments revealed priority effects during root microbiota assembly, where established communities are resilient to invasion by latecomers, and that host preference of commensal bacteria confers a competitive advantage in their cognate host. Our findings show that host preference in commensal bacteria from diverse taxonomic groups is associated with their invasiveness into standing root-associated communities.