Root colonization by beneficial rhizobacteria

Abstract Rhizosphere microbes play critical roles for plant’s growth and health. Among them, the beneficial rhizobacteria have the potential to be developed as the biofertilizer or bioinoculants for sustaining the agricultural development. The efficient rhizosphere colonization of these rhizobacteria is a prerequisite for exerting their plant beneficial functions, but the colonizing process and underlying mechanisms have not been thoroughly reviewed, especially for the nonsymbiotic beneficial rhizobacteria. This review systematically analyzed the root colonizing process of the nonsymbiotic rhizobacteria and compared it with that of the symbiotic and pathogenic bacteria. This review also highlighted the approaches to improve the root colonization efficiency and proposed to study the rhizobacterial colonization from a holistic perspective of the rhizosphere microbiome under more natural conditions.


Introduction
The significance of plant-and animal-associated microbiomes to their hosts has been well recognized for decades (Mendes et al. 2013 ).Microbes inhabiting the rhizosphere are critical determinants of plant growth and health.Beneficial rhizobacteria show great potential in agricultural production since they offer a variety of beneficial functions for plants, such as promoting plant growth and enhancing plant abiotic stress tolerance by secreting phytohormones and some specific signaling molecules and protecting host plants by inducing systemic resistance and direct antagonism with soil-borne pathogens (Pieterse et al. 2014 ).These beneficial bacteria can gener all y be used in a gricultur e as biofertilizers or microbial agents and are essential in green agricultural pr oduction.Rhizospher e colonization is one of the most important features of rhizobacteria that determines their survival and pr opa gation, whic h ar e pr er equisites for v ersatile bacteria to exert their beneficial functions on host plants (Mendes et al. 2013 ).
The rhizosphere includes plant roots and the surrounding soil influenced by root exudates (Dessaux et al. 2016 ), therefore, bacteria surviving and forming firmly community in rhizosphere soil, on rhizoplane and in root endosphere were all defined as the term "rhizosphere colonization" (Fig. 1 ).The y can selecti v el y colonize distinctiv el y on primary root or later al r oot, on spatial axis of the r oot, inside r oot, or r oot surface.Rhizobacteria colonize the plant root in a highly heterogeneous manner, covering 10%-40% of the root surface (Danhorn and Fuqua 2007 ), and some endophytic bacteria can also live inside root tissue.Since the colonization process of symbiotic bacteria, which reside in living plant cells or is surrounded by a membrane compartment (Reinhold-Hurek and Hurek 2011 ), has been thoroughly reviewed (Roy et al. 2020, Soyano et al. 2021, Yang et al. 2022, González-Guerr er o et al. 2023, Jain et al. 2023, Rahmat et al. 2023, Xu and Wang 2023 ), this r e vie w only focuses on the root colonization of nonsymbiotic beneficial rhizobacteria.
Plants are the major players in the rhizosphere and they affect bacterial colonization.Plants secrete 11%-40% of photosynthesis products into the rhizosphere as root exudates (Zhalnina et al. 2018, Du et al. 2021 ), which cause the rhizosphere to be a highly active site for microbial colonization than bulk soil.Undoubtedly, the colonization of beneficial rhizobacteria is lar gel y impacted by the abundance and composition of root exudates.Root exudates can be divided into the low molecular weight and high molecular w eight compounds.Lo w molecular w eight compounds include sugars , organic acids , amino acids , alcohols , volatile compounds, and some secondary metabolites.The high molecular weight compounds are less diverse but yield a higher mass % of root exudates, and those compounds ar e mostl y pol ysacc harides and pr oteins (Cha gas et al. 2018 ).Although the rhizosphere is rich in carbon resources for bacterial growth, it is generally accepted that plants are able to expel unfavorable bacteria through the plant immune system, which is also a crucial factor that determines bacterial colonization in the rhizosphere (Shu et al. 2023 ).The concept of plant immunity has been well-established in interactions with pathogens and symbiotic microbes.Recently, the importance of plant immunity in modulating nonsymbiotic rhizobacteria colonization has been fully recognized (Shu Figure 1.Rhizosphere colonization process of nonsymbiotic bacteria.Rhizosphere consists of the roots and the surrounding soil, and the rhizosphere colonization includes rhizosphere soil-, rhizoplane-and endophytic colonization.According to different bacterial species, the colonization process can be divided into se v er al steps, including chemotaxis and motility, root surface attachment, growth and rhizoplane biofilm formation, and endophytic penetration.Chemotaxis and motility determine the moving to w ar d rhizosphere, the initial site selection, and migration of colonization site.Attachment to the root surface is follo w ed, during which the bacteria must overcome plant immunity.Bacterial growth using root exudates as the carbon resources and competing scarce elements in rhizosphere is necessary for biofilm formation, which is required by most rhizosphere soil and rhizoplane colonizing bacteria.Endophytic bacterial species penetrate intercellular spaces within root tissue through unique mechanisms after root attachment or biofilm formation. et al. 2023 ).Additionally, a "cry-for-help" theory proposed that a stressed plant can recruit beneficial bacteria to colonize the rhizosphere (Lebeis et al. 2015, Rolfe et al. 2019 ).All these factors influence the rhizosphere colonization of the nonsymbiotic beneficial bacteria.
The biology of root colonization by rhizobacteria has advanced in r ecent years.Rhizospher e colonization is a complex pr ocess involving se v er al ste ps that de pend on bacterial lifestyles .T hey can colonize in rhizosphere soil, on rhizoplane, or endophytically based on some of these steps (Fig. 1 ).In general, rhizobacteria colonize the root in a sequential process that begins with rhizosphere c hemotaxis, r oot attac hment, sometimes follo w ed b y rhizoplane biofilm formation or endophytic colonization for some strains.Bacterial chemotactic motility involves a conserved intracellular signal transduction pathway and varied signal sensors and drives the selection of initial sites for attachment and colonization site migr ation, whic h v ary depending on the str ain and plant species (Sampedro et al. 2015, Li et al. 2022 ).After moving to the rhizospher e, some bacterial str ains need to stop moving and adhere to the root surface, which is defined as root attachment (Knights et al. 2021 ).During this period, bacteria must exert their role to overcome the plant immune response for further colonization.Rapid pr olifer ation using r oot exudates as the main carbon resources is one of most important process for colonization.Some of the rhizobacteria formed biofilm on the rhizoplane in a multispecies manner (Beauregard et al. 2013 ).During this period, bacteria have to compete for some scarce elements in the rhizosphere to support pr olifer ation and biofilm formation (Liu et al. 2023 ).Additionally, some endophytes begin penetrating into plant tissue during life on the root surface (Dudeja et al. 2021, Mushtaq et al. 2023 ).In gener al, these pr ocesses involv e complicated lifestyle tr ansformation and intracellular signal transduction that are influenced by plants and the environment.Ho w ever, the current understanding of bacterial colonization in the rhizosphere is scatter ed, especiall y for beneficial nonsymbiotic rhizobacteria.
In this r e vie w, w e will summarize the kno wledge on the rhizosphere colonization of nonsymbiotic beneficial bacteria along with the sequential process and conclude the underlying regulatory molecular mechanism, the important bacterial genes involved in the processes, and the influencing factors.We will also r e vie w the advances in "cry-for-help" theory.The difference in colonization processes and the plant −microbe interactions that determine colonization between nonsymbiotic bacteria will be compared with that of symbiotic/pathogenic bacteria.Finally, we propose se v er al artificial str ategies to enhance the colonization of beneficial rhizobacteria, which would benefit the application of beneficial rhizobacteria in a gricultur e .T he scope of this r e vie w is compr ehensiv el y summarizing the rhizosphere colonization processes of the nonsymbiotic bacteria to promote the application of beneficial rhizobacteria in a gricultur e.

Chemotaxis and motility
Chemotaxis is a motility-based ability of microbes to sense chemical gradients and direct their movement either up the gradient tow ar d the source (attraction) or down the gradient a wa y from the source (repulsion).Motility and chemotaxis of vegetative bacterial cells are essential for rhizosphere colonization, as well as for establishing primary bacteria-r oot inter actions (Feng et al. 2021a ).Root exudates activate chemosensory pathways and cause motile bacteria to move to w ar d the root.Rhizobacterial motility can be ac hie v ed by v arious mec hanisms, including fla gellar swimming, swarming, twitching, and gliding motility (Kearns 2010 ).Bacterial swimming is ac hie v ed by r otating fla gella to generate a force that moves the cell forw ar d (Sampedro et al. 2015 ).Swarming is a multicellular mo vement o ver a solid surface that is driven by a raftlike flagellar complex from the community (Kearns 2010 ).Twitching is a motility based on the extension-tethering-r etr actionextension of type IV pili (Sampedro et al. 2015 ).Gliding motility is a definition of cells moving smoothly along their long axis in the absence of any visible organelle (Mignot 2007 ).
Chemotaxis and motility then drive the selection of the initial contact site on the root.The success of these processes determines the root colonization efficiency.It is evident that either inactivation of chemosensory activity by knocking out all the c hemotaxis r eceptors or bloc king motility by deleting the genes responsible for synthesizing flagellin in a rhizobacterium led to a 100-fold decrease in root colonization efficiency (Feng et al. 2018, Tzipile vic h et al. 2021 ).

Chemotaxis process and signaling
Chemotaxis intracellular signaling is conserved in many bacterial species.Bacterial chemotaxis to w ar d root exudates is initiated b y the per ception of c hemoeffectors in r oot exudates by bacterial tr ansmembr ane c hemotaxis r eceptors, whic h ar e specificall y termed methyl-accepting chemotaxis proteins (MCPs) (Feng et al. 2021a ).Gener all y, c hemotaxis r eceptor pr oteins al ways exist in a ternary complex with the CheA histidine kinase and the coupling protein CheW.Chemotaxis receptors are transmembrane proteins that constitute a highly varied ligand-binding domain (LBD) in the extracellular space for signal sensing, an intracellular highly conserved methyl-accepting (MA) domain for adaptation, which is the standard criterion for the annotation of proteins as MCPs (Sampedro et al. 2015 ).The MCPs selectiv el y r ecognize and bind to specific ligands, such as root exudates, resulting in molecular signals that transduce across the cellular membrane .T his transduction subsequently modulates the autophosphorylation rate of the histidine kinase CheA in a CheW-dependent manner (Lacal et al. 2010 ).CheA and CheY constitute a two-component system.The phosphorylation of CheA affects the transphosphorylation of the CheY r esponse r egulator.Phosphorylated CheY binds to motor proteins that are responsible for driving various kinds of motility in different bacteria.In addition, the turnover of methylation and demethylation of the MA domain of the MCPs was deployed as an adaptation system, and methylation increased while demethylation decreased the autophosphorylation activity of CheA (Sampedro et al. 2015 ).This whole signaling pathway is extr emel y wellconserved in many bacteria, including Escherichia coli , Bacillus spp.and Pseudomonas spp.
The variety of MCPs with different LBDs determines the molecules to be sensed by the bacteria (Sanchis- López et al. 2021 ).In rhizobacteria, an expansive array of MCPs and their corresponding ligands have been identified, with notable examples found in species such as Pseudomonas putida , Bacillus velezensis , and Sinorhizobium meliloti .Allard-Massicotte et al. ( 2016 ) demonstrated that root colonization of Bacillus subtilis involves multiple c hemotaxis r eceptors.An efficient colonizer in the rhizospher e should respond to a broad range of compounds in root exudates.For example, the colonization of P. putida KT2440 and B. velezensis SQR9 was regulated by various compounds in root exudates (Ortega et al. 2017, Feng et al. 2019 ).Notably, Pseudomonas spp.exhibit c hemotactic r esponses to an impr essiv e r epertoir e of ov er 140 compounds, thereby setting them as exemplary models for elucidating the structure −function relationships between MCPs (Sampedro et al. 2015 ).A comprehensive analysis revealed that P. putida KT2440 harbors as many as 27 distinct MCPs (Corral-Lugo et al. 2016 ), each specific to detect a myriad of signaling molecules , including polyamines , amino acids , fatty acids , sugars , and many secondary metabolites.Bacillus velezensis SQR9 is endo w ed with eight unique MCPs, explicitly enumerated as McpA, McpB, McpC, McpR, TlpA, TlpB, YfmS, and HemAT (Liu et al. 2020b ).
Ho w e v er, the functions of homologous MCPs can be different between strains.For example, McpA in B. velezensis SQR9 orchestr ates c hemoattr action to a wide r ange of 20 ligands, including organic acids , sugars , and amino acids (F eng et al. 2019 ).Its homologs in B. subtilis NCIB 3610 ar e pr edominantl y pr edisposed to sugar ligands, specifically glucose and α-methylglucoside (Allard-Massicotte et al. 2016 ).Through rigorous molecular investigations coupled with site-directed mutagenesis experiments, it has been elucidated that McpA in strain SQR9 boasts a broad ligand-sensing ca pacity arising fr om its ca pability to harness both the distal and pr oximal membr ane r egions of its LBD.(Feng et al. 2022 ).Rootsecreted glucose can act as a chemoattractant to many beneficial rhizobacteria (Feng et al. 2019, Sánchez-Gil et al. 2023 ).Cucumber r oot-secr eted d -galactose serv es as a ligand of McpA in str ain SQR9 to enhance chemotaxis (Liu et al. 2020b ).Compounds that act as chemoeffectors in root exudates are mainly low molecular weight compounds, such as organic acids , amino acids , sugars , sugar alcohols, and flavonoids.Some of these compounds also act as repellents.Detailed MCPs and their sensed root exudate compounds have been summarized by Feng et al. ( 2021a ).
In addition to acting as a chemoeffector attracting bacteria, a range of compounds in root exudates enhance the motility of rhizobacteria.Root-secr eted sucr ose activ ates the bacterial pr oduction of extracellular polymeric levan, which in turn regulates the flagellar synthesis of B. subtilis , and B. subtilis cannot effectiv el y colonize roots of Arabidopsis mutants that are deficient in root sucr ose secr etion (Tian et al. 2021b ).Inter estingl y, Bacillus -pr oduced surfactin, an antibiotic essential for bacterial motility and thus rhizosphere colonization, is also promoted by other root exudates, suc h as pol ysacc harides (Debois et al. 2015, Hoff et al. 2021 ).Recent studies r e v ealed that r oot-secr eted inositol can act as a signaling molecule to stimulate swimming motility in Pseudomonas via inositol-induced r epr ession of DksA, a transcriptional regulator involved in inhibiting swimming motility and thus chemotaxis to the rhizosphere (Vílchez et al. 2020, O'Banion et al. 2023, Sánchez-Gil et al. 2023 ).The Arabidopsis r oot-secr eted flavonoids attract Aeromonas sp.H1 by upregulating transcripts of flagellum biogenesis and inhibiting fumar ate r eduction for smooth swims (He et al. 2022 ).
Notabl y, the diffusion r ange of r oot exudates is inher entl y limited, leading to reduced concentrations at greater distances from the root.In light of emerging theories on bacterial chemotaxis, ther e a ppears to be a sophisticated relay of chemotactic signals between distinct bacterial cells (Cremer et al. 2019, Insall et al. 2022 ).Although they have not identified the signaling molecules secreted by the bacteria yet (Cremer et al. 2019 ), it supports that bacterial self-generated chemotactic signals might be essential in facilitating movement to the rhizosphere.Besides by sensing self-produced signal, bacterial chemotaxis may also be achieved thr ough micr obe-micr obe inter actions (Tian et al. 2021a ), sometimes e v en by attr action to the exudates of root-associated fungi (Jiang et al. 2021, Mesny et al. 2023 ).To encapsulate this dynamic, microbes near the roots will sense root-secreted chemotactic signals and secrete chemotactic cues from their locus .T his results in the establishment of a secondary chemotactic signal gradient, effectiv el y dr awing in mor e bacterial cells and mediating bacterial advancement to w ar d the roots.

Colonization site selection and migration
Bacterial chemotaxis and motility determine colonization site selection and migration.The colonization sites can differ between bacteria, e v en between phylogenetically close strains (Fan et al. 2012, Gao et al. 2013, Tovi et al. 2019, O'Neal et al. 2020 ).It can be expected that sites with high exudation are possible colonization hotspots for the whole community because the high concentration of root exudates would attract bacteria (Darrah 1991, Marschner et al. 2011 ).Root hairs promote plants to allocate more carbon to root exudates (Holz et al. 2018 ), but it is gener all y a gr eed that the exudation rates are high in the elongation zone just behind the root tips rather than in the mature root zones .T he colonization site is tempor all y c hanged along the r oot axis or between differ ent r oot br anc hes during the colonizing life cycle (Trivedi et al. 2020 ).The long-term colonization site may be different from the initial contact site.For instance, Bacillus megaterium NCT-2 cells wer e mostl y distributed in the epidermis of the root elongation zone of maize at 3 days postinoculation (dpi), while colonization was observed along the meristematic zone, elongation zone, and root hair region at 11 dpi (Chu et al. 2018 ).
First, bacterial chemotaxis and motility contribute decisively to the selection of the initial site for colonization.O'Neal et al. ( 2020 ) found that the Azospirillum brasilense mutant lacking the major c hemor eceptors that ar e r esponsible for r oot exudate c hemotaxis is impaired in preferentially accumulating on rhizoplane and inside tissue of maturation and elongation zones .T he factors influencing bacterial chemotaxis and motility for selecting root colonization sites are complex, including the diversity and concentr ation of eac h component in r oot exudates at differ ent sites and the immune response of different cell types and some locally secr eted antimicr obial compounds (Frösc hel et al. 2021, Verbon et al. 2023 ).For example, r eactiv e oxygen species (ROS) pr oduced by roots also act as repellents to drive bacterial repulsion from the wheat root tip for initial colonization (O'Neal et al. 2020 ).Second, in addition to having a decisive role in the initial contact site, bacterial chemotaxis and motility also drive colonization site migration after root surface attachment.Root cell development c hanges the r oot exudation site, and bacterial migration could follow the c hanged r oot exudation sites, which are dynamically moving, following the expansion of the root system (Zboralski and Filion 2020 ).The migration of bacterial colonization site on roots after initial colonization can also result from evasion of immuneactiv ating sites.Spatiotempor al r oot imm une r esponses during micr obial colonization ar e an important factor that determines the bacterial colonization site (Tsai et al. 2023 ).Liu et al. ( 2018 ) suggested that the morA mutant of Pseudomonas is a poor rhizosphere colonizer due to its inability to mov e fr om the initial site of colonization after triggering plant immune responses, indicating that migration along the root may occur to e v ade plant defense after initial colonization.
Ov er all, ther e is no doubt that bacterial chemotaxis and motility determine the site pr efer ences for colonization in different root zones.Ho w e v er, most of the current research measuring rhizosphere colonization is mainly based on overall quantitative measur ements, while measur ements of colonization in differ ent r oot zones ar e lac king, whic h will lead to many objectively existing differences in colonization being ignored or some differences in colonization being misinter pr eted.The k e y problem for this status is the difficulty in measuring bacterial colonization within distinct root zones.Most current studies regarding colonization sites are based on microscopic observations, including fluorescence-, GUS-and FISH-based methods (Cao et al. 2023b ).These strategies can well-reflect in situ bacterial colonization, but they are not as accurate as traditional plate counting methods in quantification.Mor eov er, due to the limitation of displaying only partial root zones under the microscope, it can sometimes be influenced by subjective bias.

Root surface a ttac hment and interaction with plant immunity
Root surface colonization begins immediately after chemotaxis to w ar d root, with successful adhesion to the root being the critical step for rhizoplane and endophytic colonization.In brief, bacteria need to stop moving and bind to the root surface, in which a transformation of lifestyle processes controlled by complex signal transduction is involved.Comprehensive studies on representative rhizobacteria, including Pseudomonas , Bacillus , Bradyrhizobium , Azospirillum , Agrobacterium , and Salmonella , ha ve un veiled the molecular intricacies of root attachment.It has been established that a gricultur all y important micr obial species shar e a biphasic model for root attachment (Wheatley andPoole 2018 , Knights et al. 2021 ).In most cases, this biphasic model inv olves tw o steps: initial attachment occurs when rhizobacteria are reversibly bound to a root surface, follo w ed b y secondary attachment that results in their irr e v ersible attac hment (Knights et al. 2021 ).The curr ent knowledge on r oot attac hment based on these two steps will be r e vie wed her e.In addition, upon attac hment to the r oot surface, plant immunity functions as an important factor influencing bacterial colonization, which will also be discussed for the strategies bacteria deplo y ed to addr ess plant imm unity.

Reversible initial attachment
In general, initial attachment is weak, reversible, and nonspecific, allowing single cells to attach to the root surface.Compared to later-stage secondary attachment, the initial attachment is relativ el y poorl y c har acterized.Numer ous physioc hemical and electrostatic forces influence the initial interactions between the surface molecules of the root and bacterial cell en velope , including van der Waals forces, electrostatic forces, and hydrophobic interactions .To o v ercome these r e pulsi ve forces, rhizobacteria use adhesiv e pili (T-pili), fla gella, the polar flagellum, and fimbriae to ov ercome the electr ostatic r epulsion that occurs between negativ el y c har ged cell env elopes and r oot surfaces (Berne et al. 2015, Knights et al. 2021 ).For instance, the flagella-deficient mutant of A. brasilense is unable to adhere to wheat or maize r oots.Mor eov er, the polar flagella purified from A. brasilense bind to wheat roots dir ectl y (Rossi et al. 2016 ).In addition to this universal force of attachment, rhizobacteria can exhibit numerous species-specific mechanisms for attachment and colonization.The major membrane porin, outer membrane proteins, and polysaccharides are considered to play a role in root adhesion during the early stages of root establishment (Berne et al. 2015 ).An outer membrane porin F (OprF) from Pseudomonas shows adhesiv e pr operties tow ar d the roots of cucumbers and tomatoes.It was found that OprF-deficient mutants of P. fluorescens are significantly less capable of loosely adhering to roots than wild-type plants, which indicates that OprF plays an important role in primary attachment (Alv ar ez Cr espo and Valv erde 2009 ).Although OprF in Pseudomonas appears to play a role in initial attachment, its molecular mec hanism r emains unclear.

Irr eversible secondar y attachment
In the following stages of initial bacterial attac hment, onl y a small percentage of rhizobacteria switch to a stronger, more specific binding mode and generate extracellular fibrils that facilitate bacterial accumulation and aggregation, called secondary attachment (Wheatley and Poole 2018 ).A range of species-specific str ategies ar e emplo y ed b y rhizobacteria for secondary attachment.Pseudomonas spp.secrete a Ca 2 + -binding protein, LapA, via ATP-binding cassette transporters .T his protein loosely associates with bacterial surfaces, facilitating interactions with the root surface (Hinsa et al. 2003 ).LapA of P. putida is also necessary for attachment to abiotic surfaces and to plant seeds (Espinosa-Urgel et al. 2000 ).Notably , P .fluorescens m utants lac king La pA exhibit diminished initial attachment to abiotic surfaces and compromised biofilm formation abilities .T he O-antigenic chains of Pseudomonas spp.lipopol ysacc harides hav e also been linked to root attachment in cr ops suc h as tomatoes and potatoes (Spiers and Rainey 2005 ).Zhao et al. ( 2016 ) demonstrated that collagen-like proteins of B. velezensis FZB42 are critical for r oot attac hment.Recentl y, Huang et al. ( 2022 ) demonstrated that the wall teichoic acid, flagellar protein FliD, and YhaN (a putative ABC transporter) of B. velezensis SQR9 function as adhesins on both cucumber root surfaces and abiotic surfaces and are involved in rhizosphere colonization (Huang et al. 2022 ).Cyclic di-AMP, a common bacterial second messenger, influences the formation of biofilms and plant root attachments in B. subtilis (Townsley et al. 2018 ).T hese in vestigations underscor e that r oot attac hment mec hanisms ar e pivotal for successful rhizosphere colonization by bacteria.

Interaction with plant immunity
Plant immunity is one of the barriers that rhizobacteria must overcome during attachment to the root surface .T he first process depends on r ecognizing highl y conserv ed micr obe-associated molecular patterns (MAMPs), including flg22, c hitin, peptidogl ycan, and lipopol ysacc haride, by pattern r ecognition r eceptors (PRRs) and activating pattern-triggered immunity (PTI), which forms a primary defense against microbial colonization.The second layer of plant immunity is referred to as effector-triggered imm unity.Plants hav e e volv ed nucleotide binding and oligomerization domain-lik e rece ptors, which sense microbial effectors either dir ectl y or thr ough effector-induced modifications of host structures (Wang et al. 2022b ).H + /Ca 2 + ion fluxes and bursts of R OS are tw o typical cellular responses occurring within minutes after immune signaling responses.Other responses include triggering downstream defense-related gene activation, defense hormone regulation, callose deposition, camalexin biosynthesis, and antimicr obial compound accum ulation.This local imm une r esponse is always accompanied by growth inhibition as a result of the growth-defense trade-off (Liu et al. 2013 ).In addition to triggering the local immune response, beneficial rhizobacteria can also elicit the induction of systemic resistance (ISR) (Pieterse et al. 2014 ).
Evidence show that at least the PTI is engaged and influences root colonization by beneficial rhizobacteria (Yu et al. 2019b ).A recent study demonstrated that the Arabidopsis root bacterial community is involved in PTI regulation, and a group of robust, taxonomicall y div erse PTI-inhibiting str ains that ar e efficient r oot colonizers were identified (Teixeira et al. 2021 ).In addition to facilitating the colonization of PTI-regulating bacteria themselves, both individual strains and synthetic consortia that regulate PTI can increase the ability of other beneficial bacteria to colonize roots (Ma et al. 2021, Teixeira et al. 2021 ).This suggests that the interaction with plant imm unity highl y influences the root colonization of beneficial rhizobacteria.

Suppressing the root immune response
Incr easing e vidence demonstr ates that beneficial rhizobacteria can avoid being detected by root receptors that elicit immune responses, whic h ar e negativ e for bacterial colonization and plant growth.One aspect is the variation in the MAMPs, which is evidenced by the variation in flg22, one of the well-studied MAMPs.Colaianni et al. ( 2021 ) sho w ed that most of the flg22 peptide variants from beneficial bacteria failed to activate PRR FLS2 (64%) and did not significantly inhibit plant host growth (80%), suggesting no activation of an energy-costly immune response .T his kind of flg22 peptide variant altered PTI signaling output by interfering with coreceptor enlistment and by another, unidentified mechanism that triggered the typical ROS response, resulting in modulation of plant immunity (Colaianni et al. 2021 ).This finding suggests that beneficial rhizobacteria may avoid eliciting the root imm une r esponse b y deplo ying flagella with lo w immunogenic sequences to facilitate rhizosphere colonization.The advantages of a low-imm une-r esponse-eliciting fla gellin also driv e the e volution of bacterial flagellar sequences with a trade-off of motility (Parys et al. 2021 ).In addition, ther e ar e beneficial rhizobacteria that possess immunogenic MAMPs that ar e v ery similar to those of pathogens .T hey ha ve , therefore , evolved the ability to evade PRR recognition by inhibiting the interaction of their MAMP with PRRs, including through modification of the MAMP epitope, inhibition of the biosynthesis of MAMP-containing molecules, or alteration of microbial cell wall compositions (Yu et al. 2019b ).In contrast to the phytopathogen Pseudomonas syringae , which suppresses the root immune response by producing the low molecular weight phytotoxin COR, the beneficial rhizobacterium Pseudomonas suppresses the flg22-triggered immune response without producing COR (Millet et al. 2010 ).Instead, Yu et al. ( 2019a ) demonstrated that Pseudomonas capeferrum WCS358 reduces the rhizosphere pH by producing gluconic acid and its deri vati ve 2keto gluconic acid, ther efor e inhibiting the flg22-binding activity of FLS2, which requires a neutral pH en vironment.T he inhibition of FLS2 activity further suppresses the flg22-mediated o xidati ve burst and root immunity, thereby facilitating colonization (Yu et al. 2019a ).Similarly, the beneficial B. subtilis FB17 can suppr ess flg22-induced earl y r oot imm une r esponses in Arabidopsis by releasing an unidentified low molecular weight compound, whic h contr ols the J A signaling components J AR1, JIN1, and MYC2 (Lakshmanan et al. 2012 ).This suggests that beneficial rhizobacteria activ el y interfer e with plant imm une signaling by deliv ering imm une-suppr essiv e compounds.Ho w e v er, curr ent knowledge on suppressing PTI is mainly aimed at flg22, and more efforts aimed at other MAMPs on a large scale should be made to r e v eal imm une suppr ession by beneficial rhizobacteria during colonization.

Tolerance of root immune response
Once plant immunity is activated, some beneficial rhizobacteria can also utilize strategies to address the activated immune response .T he root cell-type-specific transcriptome in response to a beneficial rhizobacterium Pseudomonas simiae WCS417 r e v ealed a spatial difference in immune activation of root hairs, cortex and endodermal barrier during colonization of this strain, suggesting that a spatial selection of the colonization site would benefit immune response evasion (Verbon et al. 2023 ).A genome-wide scr een in rhizospher e Pseudomonas identified two genes, morA and spuC, that are essential in rhizosphere colonization, and the authors speculated that these two genes may confer the bacterium an ability to disperse from the initial site of colonization after triggering plant immune responses (Liu et al. 2018 ).This case proposed a potential bacterial strategy that evades root immunity through spatial mitigation of the colonization site.In addition to spatial e v asion, higher toler ance is another str ategy to addr ess the activ ated r oot imm une r esponse, suc h as the ROS burst.Recentl y, Song et al. ( 2021 )  ).The beneficial rhizobacterium B. velezensis SQR9 possesses a specific two-component regulatory system (TCS), ResDE, to tolerate the ROS produced during the flg22-triggered root imm une r esponse, whic h pr omotes rhizospher e colonization of this strain (Zhang et al. 2021 ).
Ho w e v er, it is still unclear whether the suppression of PTI in roots by beneficial rhizobacteria increases the risk of root infection by soil-borne pathogens.From the results reported by Ma et al. ( 2021 ), it seems that suppression of root PTI by beneficial rhizobacteria renders plants more susceptible to opportunistic Pseudomonas pathogens .Moreo ver, beneficial rhizobacteria can stimulate ISR, but the plant immune system actively or passively overlooks colonization by beneficial rhizobacteria during interactions.Whether this resistance impacts the colonization of nonsymbiotic beneficial rhizobacteria and its relationship with local plant immunity is unclear.

Bacterial gr o wth and biofilm forma tion
In the rhizosphere, bacterial growth using root exudates as carbon resources is an important factor influencing root colonization.In addition to carbon resources, some scarce elements, such as phosphorus and ir on, ar e also factors limiting the colonization of bacteria.Many bacterial species have evolved fascinating strategies to compete for scarce elements .Moreo ver, biofilm formation is an important process for many rhizoplane-colonizing bacterial species, motile flagellated bacterial cells differentiate into matrix-pr oducing cells, whic h stop a gglutinating, begin and form extr acellular matrix surr ounding c hains (Karygianni et al. 2020 ).The biofilm matrix binds cells and imparts many k e y features to the biofilm, and ther efor e rhizospher e colonization (Flemming et al. 2023 ).The biofilms in rhizospher e ar e gener all y formed by bacteria from multispecies, and the matrix provides a spatial structure and multiple levels of protection for the community within biofilm.

Bacterial growth using root exudates
Bacterial growth and nutrition are the most important factors influencing bacterial colonization in the rhizosphere (López et al. 2023 ), and root exudate compounds can serve as nutrients that support bacterial colonization.The ability to utilize nutrients in root exudates is critical for rhizobacteria to occupy rhizosphere nic hes.Sugars and or ganic acids constitute a lar ge fr action of exudates and are the major carbon sources for rhizobacteria (Sasse et al. 2018, Korenblum et al. 2022 ); some root-sourced VOCs, such as terpenes, can also act as nutrient sources (Schulz-Bohm et al. 2018 ).Plant root exudate nutrients can selectiv el y pr omote the colonization of specific bacteria (Wang et al. 2022a ).For instance, Huang et al. ( 2019 ) discov er ed that the specialized triterpenes thalianin, thalianyl fatty acid esters, and arabidin in root exudates of Arabidopsis modulate the r oot micr obiota by enhancing or inhibiting specific bacterial growth.Rhizobacteria that can selectiv el y metabolize certain triterpenes as carbon sources for growth hav e mor e efficient rhizospher e colonization.The r oot-secr eted compound 1-aminocyclopropane-1-carboxylic acid (ACC), which is the precursor of ethylene, can be used only by bacteria with ACC deaminase .T hese bacteria can degrade ACC as a nitrogen source, giving them a significant adv anta ge in rhizosphere colonization (Li et al. 2019 ).Recentl y, se v er al publications demonstrated that plant secreted inositol as a nutrient is important for regulating rhizobacteria colonization (O'Banion et al. 2023 ), and a conserved inositol metabolism cluster in root Pseudomonas contributes to the competition for nutrients in the rhizosphere (Sánchez-Gil et al. 2023 ).In addition to the direct effect, compounds in root exudates can be degraded by specific bacteria, and the resulting metabolites will promote colonization by other bacteria.This kind of effect is expected to gr eatl y participate in modulating root colonization by beneficial rhizobacteria (Sasse et al. 2018 ).
Some broad-spectrum antimicrobial substances in root exudates also impact the colonization of beneficial rhizobacteria by serving as carbon r esources.Man y plant secondary metabolites and small peptides exert variable antimicrobial activity (Chagas et al. 2018 ) and function as biopr otectants a gainst pathogens.Howe v er, some of these compounds have selective antimicrobial activity and can act as carbon resources for certain beneficial rhizobacteria.Rhizobacteria that can metabolize r oot-secr eted antimicr obial substances will have higher rhizosphere colonization efficiency and succeed in r oot colonization.The r oot-secr eted toxic compounds camalexin and benzoxazinoids, which are signatures of the root immune response, also promoted colonization by beneficial Pseudomonas (Hu et al. 2018(Hu et al. , K oprivo va et al. 2019 ) ).Many VOCs produced by roots can serve as antimicrobial compounds, suc h as ter penes and ter penoids, to inhibit pathogen gr owth, and inter estingl y, they can also promote specific beneficial rhizobacterial growth (Chagas et al. 2018, Schulz-Bohm et al. 2018 ).In addition, ar omatic compounds r eleased by r oots also mediate defense mec hanisms a gainst pathogens and attr act some micr obes by serving as carbon sources (Lattanzio et al. 2006 ).Indeed, Lebeis et al. ( 2015 ) demonstrated that salicylic acid, an aromatic signaling molecule responsible for many kind of plant defense response, can be used by some beneficial bacterial strains as a growth signal or as a carbon source.
Some specific transporters from either plants or bacteria have been suggested to be involved in the process of bacterial acquisition of root secreted carbon resource and contribute to the bacterial colonization in rhizosphere.Plants have developed active mec hanisms for r oot exudation.Numer ous studies hav e established that specific transporters located on the plasma membrane of root may be responsible for recruiting beneficial bacteria (Hennion et al. 2019 , Vives-Peris et al. 2020 ).The plant transporter ALMT1 plays a role in exudation of the malate and the gammaaminobutyric acid (GABA), which is one of the major carbon re-sources for rhizobacteria (Lakshmanan et al. 2012, 2013, Kamran et al. 2020 ).Arabidopsis amino acid transporter, LHT1, modulates P. simiae metabolism in the rhizospher e, whic h influence its colonization efficiency (Agorsor et al. 2023 ).Bacterial also deploy a r ange of tr ansporters to acquir e the r oot exudates.Using a combination of compar ativ e genomics and exometabolomics, Zhalnina et al. ( 2018) r e v ealed that the uptake of r oot-secr eted carbon r esour ces b y specific transporters of rhizobacteria determines their colonization, and a bacterium with an uptake transporter of the highly abundant nutritional compounds of root exudates will be highl y adv anta geous in rhizospher e colonization.They also found that the uptake of certain substances is highly variable among rhizobacteria (Zhalnina et al. 2018 ).Under controlled conditions, Lin et al. ( 2020 ) demonstrated that knockout of the ptsG gene encoding the main glucose transporter in Bacillus cereus C1 L led to a shar p decr ease in r oot colonization, suggesting the importance of bacterial transporter of root secreted carbon resources in bacterial colonization.

Biofilm formation
The formation of a biofilm is a way to maintain a critical cell mass in a specific location that is sufficient to initiate beneficial interactions with host plants (Flemming and Wuertz 2019 ).Biofilms incr ease r esistance to certain envir onmental str esses as well as antimicr obial toler ance, pr otection fr om pr otozoan pr edation, consortia metabolism, or the opportunity for horizontal gene transfer (Arnaouteli et al. 2021 ).The biofilm matrix consists of extracellular polymeric substances, including polysaccharides , proteins , amyloids , lipids , and extracellular DNA, as well as membrane vesicles and humic-like r efr actories (Flemming et al. 2023 ).

Global transcription factors in biofilm formation
Mature biofilm formation generally indicates successful rhizosphere colonization.Rhizobacterial biofilm formation on the root surface is a highl y r egulated pr ocess, as eac h species has its own molecular mechanism for responding to environmental cues (Trivedi et al. 2020 ).The cessation of movement and initiation of biofilm formation by beneficial rhizobacteria are typically governed by one or se v er al global tr anscriptional r egulators within the bacterium.Consequently, these two cellular decisions are always coupled.When cells opt to transition into a biofilm state, the gene transcription associated with motility and chemotaxis is sim ultaneousl y downr egulated.For example, biofilm formation by beneficial Bacillus in rhizosphere is governed b y tw o global transcription factors, Spo0A and DegU (Arnaouteli et al. 2016 , K oba yashi andIkemoto 2019 ).DegU controls both motility and biofilm formation by different phosphorylation le v els (K oba yashi and Ikemoto 2019 ).Spo0A also controls sporulation and biofilm formation by different phosphorylation levels (Xu et al. 2019a ).Pseudomonas deploys different oligomerization of the global transcriptional regulator FleQ to adjudge the decision of motility and biofilm formation (Nie et al. 2022 ).Deficiency of these global transcriptional regulators in bacteria always leads to sharply reduced rhizosphere colonization (Xu et al. 2014, 2017, Emonet et al. 2021 ), suggesting the critical role of lifestyle transitions in rhizosphere colonization.Suc h a mec hanism will pr e v ent the contr adictory coactivation of biofilm formation and motility during rhizosphere colonization.
The global transcriptional regulators that direct the shift from bacterial motility to biofilm formation respond to environmental cues, such as root exudates (Ivanova et al. 2023 ).This sen-sory mechanism is generally mediated by cell surface receptors such as histidine kinases, notably KinD in Bacillus (Liu et al. 2020a ).Upon perceiving specific rhizosphere signals, these receptors communicate with global regulatory factors in various ways depending on bacterial variations (Arnaouteli et al. 2021, Nie et al. 2022, Wang et al. 2022 ), prompting cells to initiate biofilm formation on root surfaces.Certain plant polysaccharides, the major components of the plant cell w all, w ere also shown to enhance the biofilm of B. subtilis by acting as signals for controlling the phosphorylation le v el of the master r egulator Spo0A and as carbon r esources for pr oducing the matrix exopol ysacc haride (Beaur egard et al. 2013 ).Inter estingl y, some signaling molecules induce both biofilm formation and trigger chemotaxis in beneficial rhizobacteria, such as cucumber root-secreted d -galactose, which could be induced by B. velezensis SQR9, serving as a signal for enhancing chemotaxis and biofilm formation of strain SQR9 in a McpA-dependent manner (Liu et al. 2020b ).The organic acids in the root exudates of peanut, including citric , malic , and oxalic acids, promoted bacterial biofilm formation of the beneficial rhizobacterium Burkholderia pyrrocinia strain P10 in rhizosphere (Han et al. 2023 ).In addition, the flavones in rice root exudates enhance biofilm formation of the nitrogen-fixing bacterium Gluconacetobacter diazotrophicus , and biofilm formation in turn recruits diazotrophic bacteria in the rhizosphere (Yan et al. 2022 ).While these are distinct processes in rhizosphere colonization, it can be expected that bacteria might exhibit differential responses to differ ent concentr ations of the same signaling molecule .T hus , a molecule could stimulate chemotaxis at greater distances from roots but favor biofilm formation on the root surface.Such dosedependent signaling is very common in biofilm and chemotaxis regulation among rhizobacteria.

Effect of self-produced secondary metabolites on biofilm formation
Rhizospher e micr oor ganisms can pr oduce man y secondary metabolites, which also impact biofilm formation.Root-secreted sucr ose activ ates the bacterial pr oduction of extr acellular pol ymeric le v an, whic h in turn r egulates the synthesis of surfactin and hyperflagellation of the bacterium (Tian et al. 2021b ).Inter estingl y, by causing potassium leakage, surfactin was demonstrated to be an essential signaling molecule in the establishment of biofilms and root colonization in B. subtilis NCIB3610 (Lopez et al. 2009 ).It has also been shown that another lipopeptide antibiotic, bacillomycin D, contributes to biofilm formation by facilitating iron acquisition.In B. velezensis SQR9, bacillomycin D specifically promotes transcription of the iron ABC transporter FeuABC by binding to its transcription factor, called Btr (Xu et al. 2019a ).Additionally, using a novel branched-chain fatty acid, bacillunoic acid, allows B. velezensis SQR9 to utilize a nov el br anc hed-c hain fatty acid called bacillunoic acid to establish a policing system for punishing cheaters within the biofilm community and to improve the community's fitness in a variety of conditions, including the root colonization process (Huang et al. 2023 ).Importantly, numerous studies have observed that siderophores play an important role in rhizobacterial biofilm formation of Bacillus spp.and Pseudomonas spp.sider ophor e-defectiv e m utants in differ ent PGPR str ains fail to form biofilms and are unable to competitiv el y colonize plant r oots (Pizarr o-Tobías et al. 2015, Qin et al. 2019, Singh et al. 2022a ).Owing to the complexity of secondary metabolites in the rhizospher e, ther e ar e numer ous secondary metabolites that affect the interaction between plants and rhizobacteria, which needs to be investigated further.

Multispecies biofilm in the rhizosphere
It has been recognized that multispecies biofilms, rather than single-species biofilms, are the most dominant bacterial lifestyle natur all y found in the rhizosphere, a consortium of bacterial isolates may form stronger biofilm on rhizoplane thus an enhanced colonization can be expected (Burmølle et al. 2014, Sadiq et al. 2021 ).T here ha ve been numerous recent studies that provide insight into the synergistic effects of multispecies biofilms in rhizospher e soil, r esulting in beneficial pr operties for plants.For example, a four-species biofilm consortium exhibited higher biomass than single species, as well as incr eased toler ance to envir onmental stress (Ren et al. 2015, Yang et al. 2021 ).In one particular instance, a consortium of fiv e rhizospher e nativ e bacterial isolates forms synergistic biofilms in vitro and colonizes a larger area on the root than the individual strains (Santhanam et al. 2015(Santhanam et al. , 2019 ) ). Inoculation of cucumber rhizosphere with B. velezensis could increase the colonization of resident plant-beneficial Pseudomonas stutzeri through synergic biofilm formation (Sun et al. 2022 ).Furthermor e, a study demonstr ated that a thr ee-species combination composed of Xanthomonas , Stenotrophomonas , and Microbacterium spp.sho w ed increasing biofilm production compared to their individual members and thus increasing beneficial function on Arabidopsis (Berendsen et al. 2018 ).

Competition for scarce elements for growth and biofilm formation
Because of the large number of organisms in the rhizosphere, ther e ar e ine vitable wars for limited elements, especiall y for the r elativ el y scarce nutrient elements that are essential for rhizobacterial colonization, such as phosphorus, iron, zinc, and manganese (Dennis et al. 2010 , Tsai andSchmidt 2017 ).Here, the scarce element nutrient is defined as the limited amount of this element in the rhizosphere becomes a limiting factor for bacterial growth and biofilm formation.In addition, plants also need these elements for growth, leading to fierce competition for phosphorus and iron in the rhizosphere.
Phosphorus gener all y r eacts with calcium and ma gnesium in alkaline soils or with aluminum and iron in acidic soils to be fixed, which is difficult to absorb and utilize, resulting in a low le v el of phosphorus availability for bacteria (Earth System Science Data Discussions 2017 ).Ra pid r oot absor ption and poor mobility often lead to phosphorus depletion in the rhizosphere (Ceulemans et al. 2017, Sakuraba et al. 2018 ).Soil phosphorus is divided into inorganic P (P i ) and or ganic P (P o ); inor ganic phosphorus mainly exists in the form of phosphate, and organic P is an insoluble complex formed with organic monoesters , diesters , and inositol phosphates (Turner 2008, Liu et al. 2022 ).To cope with such situations, a range of beneficial rhizobacteria secrete different phosphatases to dissolve organic phosphorus in soil and utilize the unique phosphorus transport system for uptake and utilization (Fitriatin et al. 2011 ).The general phosphorus solubilization and uptake system in rhizobacteria consists of four categories of genes, including the phosphorus regulatory transcription factor pho and the TCS phoB/phoR, transport system genes such as pit, pstA, pstB, and ugpQ, the inorganic phosphorus solubilization genes gcd, ppa, and ppx, and organic phosphate mineralization genes such as phoA and phoD (Wu et al. 2022 ).The phosphorus regulatory transcription regulator pho and the downstream TCS, which are conserved in most bacterial species, are essential in activating phosphorus solubilization and uptake genes in response to a low phosphorus envir onment.Activ ation of pho gener all y induces the expr ession of a series of downstream reactions to secrete phosphatases and or ganic acids, ther efor e miner alizing insoluble or ganic phosphates (Hulett 1996 ).In recent years, it has been reported that the constitutive phosphatase (PafA) activity expressed by Flavobacteria in the rhizosphere is stronger than that of Pseudomonas , which enables Flavobacteria to occupy unique phosphorus clearance sites in the rhizosphere and enhance the ability of phosphorus acquisition (Lidbury et al. 2021 ), making the Flavobacteria successful colonizers of the phosphorus solubilizing niche in the rhizosphere.
Iron is an indispensable element that participates in many important biological metabolic processes; in particular, bacterial biofilm formation r equir es sufficient iron (Qin et al. 2019, Xu et al. 2019a ).The total iron in soil is abundant, estimated to be 20-40 g/kg (Bo wles 1997 ); ho w e v er, most ir on is pr esent in insoluble iron oxide precipitates or insoluble high-valence forms.Iron av ailability is extr emel y low in neutr al and alkaline soils (Mor eno-Jiménez et al. 2019 ).Mor eov er, plant r oots also deploy a str ategy that takes up iron and withholds excess iron in vacuoles to restrict pathogen virulence .T herefore , soluble iron is extremely scarce for bacteria in the rhizosphere (Trapet et al. 2021 ).To incr ease their competitiv eness for ir on nutrition in the rhizospher e, most rhizobacteria produce siderophores to chelate ferric iron for colonization in rhizosphere (Stringlis et al. 2018b ).Bacterial sider ophor es can be hijacked by other bacteria to compete for iron (Gu et al. 2020 ).In addition to competition for soil iron by sider ophor es, ir on competition between rhizobacteria and plants is also a canonical battle field (Xing et al. 2021 ).It has been recently found that beneficial rhizobacteria also trade with iron resources during bacterial colonization.Bacillus velezensis SQR9 deploys the type VII secretion system to export YukE, which inserts into the plant root cell membrane to cause iron leakage to facilitate the iron nutrition and rhizosphere colonization of this strain (Liu et al. 2023 ).

Endophyte penetr a tion
Endophytic bacteria colonize the host tissue.Some endophytes can colonize roots from vertical transmission and have been reviewed on vertical transmission (Frank et al. 2017, Guo et al. 2021, Soluch et al. 2021 ).Here, we focus on the endophytic process after root attachment of the bacteria.The intercellular colonization process has been demonstrated with several model endophytes, such as Azoarcus spp ., P araburkholderia phytofirman , and Klebsiella spp.(Reinhold-Hurek et al. 2007, Turner et al. 2013 ).The k e y process is penetration into plant tissue (Hallmann 2001 ).The infection site selection and the bacterial featur es involv ed in lifestyle of root colonization are the k e y points here.

Infection site
The infection sites of rhizosphere endophytes are selective.It has been reported that many microorganisms enter plant root tissue by the following thr ee putativ e pathways: the root tip in the elongation and differentiation zone, the points where later al r oots emer ge, and the axils of emer ging or de v eloped later al r oots (Reinhold-Hur ek and Hur ek 1998, James 2000, Mushtaq et al. 2023 ).James et al. ( 2002 ) deployed a GUS-marked strain of the endophyte Herbaspirillum seropedicae , a nitrogen-fixing bacterium, to study the rhizosphere colonization site in rice .T his bacterium is most abundant on coleoptiles, lateral roots, and at the junctions of the major and later al r oots in the initial step (James et al. 2002, Balsanelli et al. 2010 ).It enters roots via cracks at the points of later al r oot emer gence and subsequentl y colonizes the intercellular spaces of roots (James et al. 2002 ).Histochemical analysis of seedlings of maize, sorghum, wheat, and rice grown in vermiculite sho w ed that strain H. seropedicae LR15 colonized inner tissues.In the early steps of the endophytic association, H. seropedicae colonized intercellular spaces of the root cortex; it then occupied the vascular tissue.Colonization was also observed in the external m ucila ginous r oot material at 8 dpi (Roncato-Maccari et al. 2003 ).Bacillus megaterium NCT-2 could penetrate into maize roots through the root tip in the elongation and differentiation zone (Chu et al. 2018 ).Compant et al. ( 2005 ) labeled Burkholderia sp.PsJN with GFP and observed the bacterial cells enriched in high numbers at the sites of lateral root emergence.Growing evidence support the idea that the endophytic colonization site is highly restricted by plant, such as by the plant immunity, the suberin, the casparian strip, and some antimicrobial metabolites in root tissues (Philippe et al. 2020, Durr et al. 2021, Fröschel et al. 2021, Kashyap et al. 2022, Verbon et al. 2023 ).

Specific features of bacterial endophytes
It seems that the decision of endophytic colonization can be distinct e v en between bacterial str ains with close phylogenetic relationships .For instance , two efficient a vocado root tip colonizers, P. alcaligenes AVO73 and P. pseudoalcaligenes AVO110, display distinct colonization sites; the latter colonizes root wounds and intercellular spaces between root epidermal cells, while the former colonizes only the root surface (Pliego et al. 2008 ).It is gener all y a gr eed that the factors influencing bacterial endophytism are complex and varied.Chen et al. ( 2020 ) explored the transcriptome profile of rice upon infection b y tw o endophyte isolates, Azoarcus olearius BH72 and Azospirillum sp.B510 and found that plants respond quite differ entl y to these two endophytes, suggesting a lar ge v ariation in molecular interactions during endophytic colonization.But knowledge on the bacterial genetic features that responsible for penetration into root tissue and intercellular lifestyle is still very limited.
Cell wall degradation is expected to be a fundamental skill of endophytic bacteria, e v en if they do not need to enter the intr acellular space .T he secr etion of cell wall-degr ading enzymes, mainl y pectinases and cellulases, is known to be involved in bacterial penetration into plant tissue (Compant et al. 2005 ).A mutant of A. olearius BH72 devoid of endoglucanase activity had a decreased ability to colonize rice (Reinhold-Hurek et al. 2006 ).Rat et al. ( 2021 ) tested 197 endophytic bacteria of medicinal plant Alkanna tinctoria and found strains expressing cell-wall degrading enzymatic activities might have strong plant growth-promoting activity due to their ability to colonize plant.
A unique r espir atory type of metabolism may be essential for an endophyte because the carbon resources and the oxygen in plant tissue are quite different from those in the rhizoplane and soil.For example, the well-studied endophyte A. olearius BH72 has a strictl y r espir atory type of metabolism and cannot utilize common carbohydr ates (Kr ause et al. 2006 ).A highl y ada ptiv e r espir atory type can be expected to be essential for root endophytic life of bacteria.
Unique motility may function in e v ading plant tissue.Böhm et al. ( 2007 ) demonstrated that a type IV pili-dependent twitching motility, but not the type-pili itself, mediated the endophyte A. olearius BH72 invasion of and establishment inside the plant.
The interaction with plant immunity is expected to be a major trait for the ada ptiv e lifestyle of endophytes.It has been shown that a plant-beneficial endophyte gener all y elicits a weaker imm une r esponse than pathogens.Mor eov er, Deng et al. ( 2019 ) demonstrated that an endophyte B. subtilis strain could evade plant defense by producing subtilomycin to mask self-produced flg22.Activation of the immune response or other stress responses is always accompanied by o xidati v e bursts, whic h lead to osmotic stress in endophytes, so it can be expected that a successful endophyte also harbors ROS tolerance to address the plant immune response and the ROS produced by plants under stressful conditions.Alquéres et al. ( 2013 ) found that the endophyte G. diazotrophicus PAL5 sho w ed incr eased expr ession of genes encoding ROS-detoxifying enzymes during colonization in rice roots.
In conclusion, knowledge on the molecular mechanism underlying the endophytic lifestyle is still lacking.First, although the feasible and independent solutions for endophyte isolation have been demonstrated, a standardized and unbiased method is urgentl y needed.A compr ehensiv e genomic comparison will help to determine whether there is a common trait in the genome of bacterial endophytes.To identify genes involved in the endophytic lifestyle rather than contributing to the colonizing process before entering plant tissue using mutational experiments, comparing colonization both on the root surface and in root tissue is necessary.In addition, it could also be that endophytism is transient and opportunistic rather than a strict lifestyle.

"Cry-for-help" theory for root colonization of rhizobacteria
Se v er al pa pers demonstr ated that str essed plants r ecruit beneficial bacteria to colonize the r oot, ther eby facilitating the stressinduced opposite effect on plant growth and health (Berendsen et al. 2018, Yuan et al. 2018, Santo y o 2022, Xie et al. 2022, Wen et al. 2023 ).It is a noteworthy factor that influences bacterial colonization.One of the w ell-kno wn strategies is the "cry for help" hypothesis, which explains the long-term disease suppressive soil feedback to foliar pathogen attac k.The underl ying mec hanism still remain to be demonstrated (Wang and Song 2022 ).Although the current understanding of the cross-talk between root exudation, the r oot imm une system, and the "cry for help" r esponse is limited, it can be expected or confirmed that they may be linked internally.Rolfe et al. ( 2019 ) proposed three stages for this plant disease-induced long-term r esponse: r oot imm une r esponses to attac kers, str ess-induced c hanges in r oot exudation of antimicr obials and signaling chemicals, and impacts of root exudates on the rhizospher e micr obiome.In addition, e vidence has shown that root exudation from abiotic stressed plants also promotes colonization of beneficial rhizobacteria, which function to relieve the str ess r esponse of the plant.This stress-induced host selection would highly influence the colonization of beneficial rhizobacteria by changing the immune response and root exudation.

Biotic stress triggered "cry for help" response
Rudr a ppa et al. ( 2008 ) were the first to provide experimental evidence that abov egr ound disease alters root exudation of a primary root metabolite, l -malic acid, resulting in increased root colonization by a beneficial rhizobacterial strain.The authors propose that P. syringae pathovar tomato DC3000 ( Pst DC3000) infection of Arabidopsis leaves induces root secretion of l -malic acid, which acts as a specific signal for chemotaxis and colonization of the biocontrol bacterium B. subtilis FB17 in the rhizosphere (Rudrappa et al. 2008 ).A follow-up study demonstrated that either MAMPs, such as flg22, or the pathogen-deri ved phytoto xin COR are necessary to induce plants to secrete l -malic acid to promote colonization by B. subtilis FB17 (Lakshmanan et al. 2012 ).
Ho w e v er, the mec hanism that triggers the colonization promotion response is unclear.Regulation of the immune system upon perception of foliar pathogens was thought to contribute to influencing root microbiome composition (Lebeis et al. 2015 ).Foliar attack by pathogens or insects can influence belowground dir ect and indir ect plant defense r esponses (Bezemer and Van Dam 2005 ), but the root immune system needs to differentiate between beneficial and pathogenic microbes and mount appropriate, yet diametrically opposed, colonization-enabling or defense responses.Ho w ever, COR, as a mimic of JA-Ile, was proposed to suppress SA signaling and the flg22-triggered immune response (Li et al. 2005, Melotto et al. 2006 ), since both flg22 and COR could trigger the colonization pr omotion r esponse.It is ambiguous how the immune response in aboveground tissue is involved in promoting root colonization by Bacillus .It is hypothesized that some defense signaling activated upon infection by pathogen may be positive for beneficial rhizobacterial colonization.Indeed, Yang et al. ( 2023 ) found that the SA signaling pathway is essential for eliciting plants to pr omote r oot colonization of some biocontrol bacteria for bacterial wilt disease.
Another important case comes from the interaction between Fusarium and plants.Liu et al. ( 2017 ) used a split-root system to show that inoculation of part of the cucumber root system with Fusarium changes numerous root exudates and promotes colonization of the beneficial rhizobacterium B. velezensis SQR9 in distal r oots, whic h was linked to incr eased exudation of tryptophan, a biofilm formation stimulator of strain SQR9.This finding was also corr obor ated by a comics study by Wen et al. ( 2023 ), who found that Fusarium -infected cucumber also attracted Sphingomonas in addition to Bacillus for root colonization by stimulating the genes involved in motility and chemotaxis (Wen et al. 2023 ).Similarl y, Sc hulz-Bohm et al. ( 2018 ) found that upon infection with the fungal pathogen Fusarium culmorum , Carex arenaria changed the blend of root-secreted VOCs that promote the colonization of specific bacteria with antifungal properties.Root exudates from Fusarium -infected maize also stimulate root colonization of B. am yloliquef aciens OR2-30 by stimulating chemotaxis and motility (Xie et al. 2022 ).In wheat, Fusarium infection leads to higher root colonization of Stenotrophomonas rhizophila SR80, a dominant beneficial bacterium that induces strong disease resistance by boosting plant defense in abov egr ound plant parts (Liu et al. 2021 ).
Upon infection by phytopathogens, plant r oots r elease se v er al antimicrobial compounds, but little is known about their effects on root colonization by beneficial rhizobacteria.One interesting field of how these antimicrobial compounds contribute to the "cry for help" response and affect beneficial bacterial colonization is studies on the rhizosphere function of coumarin.Coumarin is a class of phenolic secondary metabolites synthesized by Arabidopsis that can stimulate biofilm formation of B. subtilis (Korenblum et al. 2022 ).Stringlis et al. ( 2018a ) r e v ealed that coumarin scopoletin selectiv el y inhibits the soil-borne fungal pathogens Fusarium oxysporum and Verticillium dahliae , while gr owth-pr omoting and resistance-inducing Pseudomonas are highly tolerant to scopoletin.Vismans et al. ( 2022 ) found that foliar infection of Arabidopsis thaliana by the biotr ophic down y milde w pathogen Hyaloperonospor a ar abidopsidis recruits beneficial bacteria that can enhance plant resistance, while it is evident that the coumarin biosynthesis genes MYB72 and F6'H1 in Arabidopsis are essential for recruiting beneficial bacterial colonization upon infection.T hese findings dra w the outline of a fascinating "cry for help" response.

Abiotic stress triggered "cry for help" response
The colonization of beneficial rhizobacteria on roots can also be activated by plants under abiotic stress.For instance, rice during and after dr ought r ecruits beneficial Streptom yces to colonize the root endosphere (Santos-Medellín et al. 2021 ).Drought typicall y decr eases the r oot exudation of plants, but dr ought-str essed tr ees hav e incr eased r oot exudation of phenolic acid compounds and quinate to recruit beneficial Bacillus and Pseudomonas for colonization (Oppenheimer-Shaanan et al. 2022 ).Root secretion of fla vonoids , whic h is often ele v ated in plants under abiotic stress , ma y also be involved in promoting colonization upon stress pr oduction.Arabidopsis r oots under dehydr ation str ess incr eased flavonoid accumulation within 15 min.The flavonoid naringenin enhances root colonization of Aeromonas sp.H1, which is identified as a plant beneficial bacterium capable of enhancing plant resistance to dehydration through transcriptional enhancement of bacterial motility and colonization (He et al. 2022 ).Hou et al. ( 2021 ) demonstrated that Arabidopsis under low photosynthesis drives the recruitment of specific rhizobacteria with beneficial effects.Plants under salt stress employ a species-specific strategy to promote colonization by beneficial bacteria in the rhizosphere.Root exudates from the salt-stressed coastal halophyte Limonium sinense promote the growth, chemotaxis and finally root colonization of the bacterium B. flexus KLBMP 4941 (Li et al. 2021d ).An interesting example is coumarins, besides mediating the pathogeninfection-triggered "cry for help" response , coumarins ha ve also demonstrated to be secreted by A. thaliana upon iron-deficiency stress to recruit beneficial bacteria (Harbort et al. 2020 ).Besides the specific molecules, stress-induced plant hormones are essential for plants to recruit beneficial bacteria.Indeed, Chen et al. ( 2020 ) found that peanut root could sense the c y anide str ess pr oduced by neighboring cassav a plants and pr oduce ethylene to recruit beneficial bacteria to adjust to the stressful environment.

Comparison with pathogenic/symbiotic bacteria for rhizosphere colonization mechanisms
Pathogenic , symbiotic , and nonsymbiotic rhizobacteria r epr esent thr ee gr oups of r oot colonizers that ar e tightl y associated with plant.But the comparison of the difference of their colonization mechanisms in rhizosphere is lack.The rhizosphere chemotaxis and root attachment of these bacterial groups are similar, whic h ar e mainl y by sensing r oot secr eted signals , mo ving tow ar d rhizosphere, and adhering to root surface, although the signals or cellular molecular pathway involved may different.The colonization process for pathogenic/symbiotic bacteria and the nonsymbiotic beneficial bacteria differed mainly in their specific lifestyles.Most nonsymbiotic rhizobacteria colonize the rhizoplane as a community, some endophytes colonize the intercellular spaces of the root at a controlled low density (Lugtenberg and Kamilova 2009 ).Ho w ever, symbiotic bacteria colonize roots intracellularly and sometimes they induce root to develop specific organs, which allow their high populations in root (Tang et al. 2020 ).Pathogenic bacteria infect root tissues and always grow to a very high density, which is needed for expression of virulence factors (von Bodman et al. 2003 ).The different lifestyles lead to difference of host specificity, nutrition and metabolism and strategies a gainst plant imm unity during colonization in the rhizosphere (Fig. 2 ).

Figure 2.
Comparison of the colonization of nonsymbiotic rhizobacteria with symbiotic and pathogenic bacteria.The r elativ e bacterial density of a nonsymbiotic rhizobacterium in its colonization site is lo w er than that of symbiotic and pathogenic bacteria.Nonsymbiotic rhizobacteria gener all y hav e br oad host r ange , while symbiotic and pathogenic bacteria ha v e v ery specific host.Symbiotic bacteria acquir e carbon r esources dir ectl y fr om the root cells and feed root cells with nitrogen, pathogenic bacteria hijack plant metabolism and nutrition, while nonsymbiotic rhizobacteria mainly use root exudates and the secretions in intercellular spaces.Symbiotic bacteria have specific interaction with plant immunity to establish infection and symbiosis, pathogenic bacteria block plant immune response by injecting effectors into root cells.

Host specificity
Gener all y, a nonsymbiotic beneficial rhizobacterium can colonize a broad range of host plants.For example, B. velezensis SQR9 was isolated from the rhizosphere of cucumber and can colonize Arabidopsis , maize and rice efficiently (Liu et al. 2014, Cao et al. 2023a ).Pseudomonas simiae WCS417 was isolated from the rhizosphere of wheat and induced systemic resistance in Arabidopsis , tomato, and many other plant species, suggesting efficient colonization of these plant species (Berendsen et al. 2015 ).The endophytes A. olearius BH72 was isolated from Kallar grass ( Leptochloa fusca L. Kunth), while it also endophytically colonized rice (Hurek and Reinhold-Hurek 2003 ).Ho w ever, relatively strict host selection is observed for symbiotic and pathogenic bacteria.Isolates belonging to Rhizobiaceae only infect legumes as a very specific host.One rhizobium strain can not colonize different cultivars from the same host plant species .T his opinion is highly supported by the r esults fr om Dong et al. ( 2021 ), who found that the legume Medicago truncatula possesses an SHR-SCR stem cell pr ogr am in cortical cells to specificall y inter act with rhizobia for nodulation.Pathogenic bacteria also have strict host selection.For example, one strain from P. syringae generally has a very limited host plant species and e v en a fe w cultiv ars fr om a single plant species, based on which the basis of the pathogenic P. syringae can be grouped into pathovars (Xin and He 2013 ).
The narrow host spectrum for symbiotic and pathogenic bacteria is gener all y due to their host selection genes, and the presence or absence of these genes determines the infection of a specific host.For example, a common concept of the presence of pathogenic bacteria and symbiotic strains is called avirulent genes, which enable specific nonhost plants to specifically prevent the infection of that strain.These avirulent genes typically mediate imm une r ecognition by nonhost plants (Yang et al. 2010 ).In contr ast, ther e ar e curr entl y no r eported host selection genes in nonsymbiotic beneficial rhizobacteria.But nonsymbiotic rhizobacteria do have a host preference, which suggest the existence of specific genes determines the colonization of these bacteria (Wippel et al. 2021 ).Ev en though, her e is curr entl y a tendency to belie v e that suc h bacteria use lo w er amplification rates in association with host plant in exchange for a wider host range.

Nutrition and metabolism
Lifestyle determines the metabolism of the bacteria.Due to the intracellular life of symbiotic bacteria, their metabolism and carbon r esources ar e lar gel y dependent on their host cells, and ther efor e, they gener all y hav e a mor e specific metabolites exc hange with the host.Intracellular colonization is established and partially controlled by plant genes.For example, rhizobia mainly use the carbohydrates of host plants as carbon resources and feed plants with ammonia during root nodule symbiosis (Yang et al. 2022 ).Moreov er, the r espir atory type and redox potential of symbiotic bacteria are highly influenced by the host plant (Yu et al. 2018 ).Specific metabolism was also observed in the well-studied Agrobacteria strategy, during which pathogenic Agrobacterium hijacks plant cells by injecting a part of the DNA sequence from the Ti plasmid to produce opines as dedicated carbon resources for Agrobacterium itself (Lang et al. 2013, González-Mula et al. 2018 , Matv ee v a and Otten 2021 ).The plant pathogen Ralstonia solanacearum is also able to manipulate plant metabolism to produce GABA to support bacterial nutrition during colonization (Xian et al. 2020 ).
The nutrition and metabolism of most nonsymbiotic rhizobacteria are not strictly dependent on the host.They mainly use a br oad r ange of or ganic compounds in r oot exudates for colonization (Badri and Vivanco 2009 ).In contrast to the specific carbon resources for bacteria during nodulation or infection, due to the m uc h higher diversity of bacteria than intercellular and intracellular spaces, the bacteria colonizing the root surface should have a broader carbon source utilization spectrum to compete for nutrients in root exudates (Mataigne et al. 2022 ).The diversity of the bacteria in the rhizosphere led them to share the various compounds of the root exudates (Yang et al. 2017(Yang et al. , 2019 ) ). Mor eov er, most nonsymbiotic rhizobacteria can degrade and use the soilderived carbon resources.

Plant immunity e v ading str a tegy
The lifestyle of pathogenic, symbiotic, and nonsymbiotic bacteria is lar gel y distinctiv e, leading a quite different strategy to interact with plant immunity.Due to the intracellular lifestyle of symbiotic bacteria, activation of the plant immune response is believed to be harmful to the interaction (Feng et al. 2021b ).Most pathogenic bacteria infect root tissue in a high density, eliciting a stressful and PAMP-ric h envir onment; when pathogens do not hav e imm unebloc king str ategies, str ong PTI and shar pl y r educed colonization can be expected (Wei et al. 2018 ).Nonsymbiotic bacteria generally colonize the rhizosphere at a relatively lo w er density, but R OS accumulation or establishment of immune response within roots has a weaker influence to the colonization of nonsymbiotic bacteria than to the pathogenic and symbiotic bacteria (Buschart et al. 2012, Zhang et al. 2021 ).T his ma y r el y on the differ ent concentr ations of antibacterial compounds, such as ROS, in root cells, intercellular spaces, and rhizoplane.The difference has been evident by se v er al studies that bloc king the plant imm une r esponse e v ading mechanism in bacteria has a much stronger impact on colonization of rhizobia and pathogenic bacteria than that of nonsymbiotic beneficial bacteria (Liang et al. 2013, Wei et al. 2015, Deng et al. 2019, Pfeilmeier et al. 2019, Yu et al. 2019a, Zhang et al. 2021 ).To fit their unique lifestyles , pathogenic , symbiotic , and nonsymbiotic bacteria deplo y ed differ ent str ategies to e v ade plant imm unity.
Pathogenic and symbiotic bacteria possess highly immunogenic MAMPs.Although many MAMPs from nonsymbiotic rhizobacteria have been identified, current researches suggest those MAMPs elicit a weaker response than that derived from pathogens, which is shown by a lower elicitation of defense gene transcription, a lo w er o xidati ve burst, and a higher concentration needed for seedling growth inhibition (Colaianni et al. 2021, Zhang et al. 2021 ).For example, Colaianni et al. ( 2021 ) demonstrated that the flg22 variant from beneficial Bacillus can not trigger seedling growth inhibition when applied to a final concentration of 10 nM, a concentration the flg22 variant from Pst DC3000 did.Ho w e v er, pathogens use unique secretion system to interfere the PTI ther efor e establishing disease (Shu et al. 2023 ).For example, both pathogenic P. syringae and R. solanacearum deliver effectors into plant cells through the type III secretion system to interfere with the plant immune response for efficient colonization (Y uan et al. 2021 , Y u et al. 2022 ).The nodulation out proteins secreted by symbiotic bacteria have been reported to suppress PTI (Xin et al. 2012 ).Both symbiotic and pathogenic bacteria show specific interactions with the plant imm une system, suc h as R genes.For rhizobia, it has also been demonstrated that R genes in legumes control the host specificity of rhizobium symbiosis.But different with pathogen, balanced regulation of innate immunity is r equir ed for rhizobial infection and symbiosis (Cao et al. 2017, Yang et al. 2022 ).In contrast, nonsymbiotic rhizobacteria regulate the plant immune response in general as reviewed in the section "Interaction with plant immunity", rather than through specific interactions as that of pathogenic bacteria and have never been shown to interact with R genes in plants.

Artificial enhancement of root colonization by beneficial rhizobacteria
The field application of beneficial rhizobacteria is an effective practice for sustainable agriculture, the efficient root colonization of these bacteria is critical for the performance of their beneficial functions .Hence , it is important to de v elop str ategies to enhance the root colonization of beneficial rhizobacteria.This r e vie w pr oposes thr ee str ategies, whic h include the addition of colonizationenhancing substrates, bacterial genetic modulation, and evolution of beneficial rhizobacteria (Fig. 3 ).
It can be expected that the application of some compounds in root exudates or microbial metabolites may serve as root colo-Figure 3. Strategies to promote rhizosphere colonization of nonsymbiotic bacteria.(A) Many compounds in rhizosphere, mainly from the root exudates , ha ve been identified to be positive signaling molecules for beneficial bacterial colonization in rhizosphere.It is a practicable way to develop such molecules as colonization stimulator and applied with the beneficial bacteria together in agriculture.(B) Many bacterial genes have been identified to be positive for rhizosphere colonization with clear mechanisms.Genetic modulation of the beneficial bacteria by introducing "colonization positive" genes would generate engineered strains as better colonizers.(C) Efficient rhizosphere colonization is a beneficial trait for bacteria itself, because rhizosphere supplied more nutrient for bacterial proliferation, therefore a continuous life in rhizosphere is expected to drive the accumulation of "colonization positive" mutations in bacterial genome.So, round-by-round inoculation and reisolation of bacteria in rhizosphere will domesticate an e volv ed str ain as a better colonizer.nization stimulators given that many studies have demonstrated the role of these compounds in modulating the root colonization of beneficial rhizobacteria.For example, the application of organic acids, such as malic acids, citric acid, and fumaric acid, can enhance root colonization of the beneficial str ains Hanssc hlegelia zhihuaiae , B. velezensis SQR9, and B. pyrrocini (Zhang et al. 2014, 2015, 2018, 2022, Feng et al. 2018, Han et al. 2023 ).Ther efor e, soil amendments can be used to promote beneficial bacterial colonization.
Genetic engineering of beneficial rhizobacteria to respond to specific root exudate compounds is another strategy to enhance colonization.Xu et al. ( 2019b ) de v eloped a xylose-inducible degQ geneticall y engineer ed str ain of B. velezensis SQR9, whic h can use r oot secr eted xylose as a signal to r egulate the phosphorylation le v el of DegU and then promoted its ability to form biofilm on the r oot surface.Compar ed to the wild-type strain, the genetically engineer ed str ain sho w ed gr eater r oot colonization ability and biocontrol efficacy in cucumber and tomato (Xu et al. 2019b ).Singh et al. ( 2022b ) engineered the beneficial bacterium A. brasilense Sp7 with enhanced d -glucose utilization ability and sho w ed significantl y incr eased r oot colonization in rice compar ed with the wildtype strain.
One ima ginativ e str ategy for impr oving r oot colonization ability of beneficial rhizobacteria is coevolution of the strain with the target plant to get the evolved strain, which is termed as targeted domestication.It is known that natural genetic m utations, suc h as random point mutation and horizontal gene tr ansfer, driv e the evolution of bacteria, for example, phage infection drive the evolution of bacterial resistance to phage (Hussain et al. 2021 ).There-fore, if a bacterial strain was inoculated to the rhizosphere, isolated and reinoculated again, then, after rounds of repeating, the genetic mutations in the evolved bacterial genome that benefit its life in rhizosphere will accumulate due to the survival of the fittest theory.It can be inferred that artificial domestication of bacterial strains within the rhizosphere under monoassociation conditions may accelerate evolution and drive the direction to a better rhizosphere colonizer.Indeed, Blake et al. ( 2021 ) found that B. subtilis NCIB 3610 differentiated into three different colony morphologies after experimental evolution within the Arabidopsis rhizosphere and that a mixture of the three morphotypes colonized the rhizosphere better than each colony alone.Li et al. ( 2021c ) repeatedly inoculated Pseudomonas protegens CHA0 in the rhizosphere of A. thaliana cultivated in sandy soil for six gro wth c ycles, and they detected 35 mutations within 28 genes in the genome of the e volv ed isolates.Among them, mutations affecting global regulators, bacterial cell surface structure, and motility accumulated in parallel acr oss m ultiple e volv ed str ains (Li et al. 2021c ).Mor eov er, the r elationship between bacteria and plants has e volv ed fr om anta gonism to mutualistic cooperation, which is manifested in a stronger ability to utilize rhizosphere exudates and a str onger toler ance to antibacterial substances secreted by plants (Li et al. 2021a ).Howe v er, the entir e tr ait corr elation networks of P. protegens CHA0 are recombined after adaptive ev olution, sho wing the loss of stress resistance modules and the linking of those modules that enhance ability after evolution (Li et al. 2021b ).Compared with the solid substr ate envir onment, domestication within the Arabidopsis rhizosphere under a hydroponic environment places more emphasis on the mobility and recolonization ability of strains (Nor dgaar d et al. 2022 ).Rotating cr oplands pr ovide a more complex ecological environment for bacteria.In an evolutionary experimental study, the e volutionary str ains in alternate host envir onments had a higher degree of parallel evolution at the gene level (Hu et al. 2023 ).Ada ptiv e m utations in B. subtilis NCIB 3610 occurr ed earlier in the presence of Pseudomonas in the rhizosphere, suggesting that a competitive environment accelerates this capacity change (Pomerleau et al. 2023 ).In conclusion, evolution experiments can be used as an important means to breed beneficial rhizobacteria with impr ov ed r oot colonization and a gricultur al a pplication.Ho w e v er, this e v olution-based domestication is also risk y because a slight environmental difference may lead to a butterfly effect on the resultant strains' features .Moreo ver, domestication of the bacteria in a simplified environment w ould w eaken the bacterial ability in other en vironments , such as stress tolerance (Li et al. 2021a , b , c ).

Conclusion and outlook
The importance of rhizobacteria in plant gr owth, de v elopment, and health has been well recognized.Recent studies have revealed many fascinating models that describe complex interactions between rhizobacteria and plant and soil en vironments .Howe v er, compared with the soil-borne pathogenic and symbiotic bacteria of rhizobia, the root colonization of beneficial rhizobacteria has not been compr ehensiv el y concluded.Her e, we summarized the root colonization of rhizobacteria into se v er al steps.We also compar ed the differ ence in the colonization pr ocess of those nonsymbiotic beneficial rhizobacteria with symbiotic and pathogenic bacteria.Finally, we discussed the efforts made to impr ov e the r oot colonization of beneficial rhizobacteria, which will facilitate their a gricultur al a pplication.
The nonsymbiotic rhizobacteria r epr esent the plant-associated bacteria with the largest abundance and diversity in the rhizosphere .T he mechanism of root colonization of nonsymbiotic bacteria is significantl y differ ent fr om that of symbiotic and pathogenic bacteria.The colonization of any nonsymbiotic strain can not reach the abundance le v el as that of symbiotic or pathogenic bacteria.The symbiotic and pathogenic bacteria colonize the inside root tissues with very high abundance, while most nonsymbiotic beneficial rhizobacteria colonize the root surface or inside root tissue with a low abundance .T he differences in colonization site and abundance suggest that the nonsymbiotic rhizobacteria hav e differ ent r oot-bacteria inter action mec hanisms.In particular, how do plants recognize nonsymbiotic beneficial rhizobacteria and allow colonization?
The rhizosphere environment is rich in other organisms, including fungi, protozoans, viruses, and other bacteria.Mor eov er, the microbiome in the rhizosphere is dominated by nonplant factors and varies largely depending on environmental factors, such as soil type, temper atur e, and humidity.Based on these concerns, the root colonization study of beneficial rhizobacteria in more natural conditions and under the holistic view of the rhizosphere micr obiome and e v en the m ultitr ophic inter action le v el will pr ovide an in-depth understanding of the process and mechanisms in the futur e. Benefiting fr om the de v elopment of sequencing tec hnology, man y studies hav e made gr eat efforts to use bioinformatic methods to analyze the rhizosphere microbiome.
Finally, the study of rhizobacterial root colonization aims to impr ov e the a gricultur al a pplication efficiency of biofertilizers, whic h ar e mostl y isolated fr om beneficial rhizobacteria.Ther efor e, our futur e study of rhizobacterial root colonization should pay more attention to the de v elopment of pr oducts or biotec hnologies based on the process and mechanism understanding to impr ov e the field application effect of beneficial rhizobacteria.More efforts to develop a new generation of biofertilizers that enhance beneficial rhizobacterial colonization should be made to promote the sustainable development of agriculture.
demonstrated that ROS in roots regulate the levels of rhizosphere beneficial Pseudomonas .The auxin produced by the beneficial bacterium B. velezensis FZB42 is essential for root colonization by antagonizing ROS produced as part of the receptor EFR-triggered immune response (Tzipilevich et al. 2021 ).Moreover, ROS induce auxin synthesis in B. velezensis FZB42 (Tzipile vic h et al. 2021