Association of rhizospheric/ endophytic bacteria with plants: A potential gateway to sustainable agriculture

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ISSN: 2276-7770
Association of
Rhizospheric/Endophytic
Bacteria with Plants: A
Potential Gateway to
Sustainable Agriculture
By
Prabhat N Jha
Garima Gupta
Prameela Jha
Rajesh Mehrotra

Greener Journal of Agricultural Sciences ISSN: 2276-7770 Vol. 3 (2), pp. 073-084, February 2013.
www.gjournals.org 73
Research Article
Association of Rhizospheric/Endophytic Bacteria with
Plants: A Potential Gateway to Sustainable
Agriculture
*Prabhat N. Jha, Garima Gupta, Prameela Jha and Rajesh Mehrotra
Department of Biological Sciences, Birla Institute of Technology and Science, Pilani-333031 Rajasthan, India
*Corresponding Author’s Email: prabhatjha@pilani.bits-pilani.ac.in Tel.: +91 1596 245073 273; Fax: +91 1596
244183.
ABSTRACT
Application of associative bacteria for sustainable agriculture holds immense potential. These bacteria are known to
enhance growth and yield of plants by fixing atmospheric nitrogen, solubilization of phosphate, production of
phytohormones and siderophores, possession of antagonistic activity as well as reducing the level of stress ethylene in
host plants. Colonization of these bacteria can be tracked by tagging them with certain molecular markers such as β-
glucuronidase (gus) or green fluorescent protein (gfp) followed by electron microscopy or laser scanning confocal
microscopy. Associative bacteria and endophytes may express genes differentially to colonize and establish the plant
interior. They may also use ‘quorum sensing’ molecules for colonization process. Present review aims to highlight
various plant growth promoting properties, ecology and updates of molecular mechanisms involved in interaction
between associative bacteria and plants as well as immune responses triggered by these bacteria in plants.
Keywords: Associative bacteria, endophyte, diazotrophy, biocontrol, induced systemic tolerance, induced systemic
resistance.
INTRODUCTION
The over increasing population of the world has already touched the number of 6.8 billion. To feed this burgeoning
population, farmers heavily rely on the use of chemical fertilizers especially inorganic nitrogen. Application of
inorganic fertilizer has many repercussions, as it leads to ground and surface water contamination due to leaching
and denitrification, which is detrimental for human and animal health. Secondly, manufacturing of industrial nitrogen
fertilizer uses non-renewable resources like natural gas and coal and causes production of green house gases viz.,
CO2 and NO2 contributing to global warming (Bhattacharjee et al., 2008). Therefore, it’s high time to opt for
alternative fertilizers which can be used in sustainable agricultural practices without affecting the environment.
Application of plant growth promoting associative bacteria can be a potential option for enhancing growth and yield of
plant in sustainable manner.
On the basis of area of colonization, Plant Associated Bacteria (PAB) can be grouped into associative
bacteria that include rhizospheric (in vicinity of root) and rhizoplanic (on surface of root) bacteria and, endophytic
bacteria. Term ‘endophytic bacteria’ is referred to those bacteria, which colonizes in the interior of the plant parts, viz,
root, stem or seeds without causing any harmful effect on host plant (Hallmann et al., 1997). These bacteria may
promote plant growth in terms of increased germination rates, biomass, leaf area, chlorophyll content, nitrogen
content, protein content, hydraulic activity, roots and shoot length, yield and tolerance to abiotic stresses like draught,
flood, salinity etc. PAB can promote plant growth directly through Biological Nitrogen Fixation (BNF), phytohormone
production, phosphate solubilization, inhibition of ethylene biosynthesis in response to biotic or abiotic
stress (induced systemic tolerance) etc., or indirectly through inducing resistance to pathogen
(Bhattacharya and Jha, 2012). Present review aims to focus on plant growth promoting abilities of rhizospheric and
endophytic bacteria and their molecular aspects. PAB has been classified as the plant growth promoting bacteria on
the basis of basic mechanisms through which it stimulates plant growth as PGPB, which induces plant growth directly
and; bio-controller, which protects plants by inhibiting growth of pathogen and/or insect (Fig. 1) (Backman and

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Sikora, 2008). In the present review, discussion regarding PGPB has excluded rhizobia associated with leguminous
plants.
Figure 1: Properties of associative/endophytic bacteria for plant growth improvement. Based on the properties,
associative/endophytic bacteria have been classified as Plant Growth Promoting Bacteria (PGPB) and biocontrol
bacteria. PGPB may benefit associated plants through providing nutrition (nitrogen, phosphorous and iron),
production of plant hormone and may enable plant tolerate abiotic stressors. Biocontrol bacteria (right panel in figure)
protect plants from invasion of pathogenic microorganisms through antagonism and/or induced systemic resistance.
Plant Growth Promoting Bacteria
Associative bacteria as well as endophytic bacteria use same mechanisms to influence plant growth. However, they
differ in efficiency through which they exert their beneficial effect. Based on various properties, plant growth
promoting bacteria can be classified as biofertilizers, rhizoremediators, phytostimulators and stress controllers.
Bacterial fertilizer is referred to the bacteria that supply nutrition to the associated plant. They may benefit plants by
providing utilizable nitrogen through fixation of atmospheric nitrogen or they make free phosphate available from
insoluble source of phosphate. Plant growth promotion due to solubilization of zinc compound driven by
Gluconoacetobacter has also been reported Beneficial properties of these bacteria are described below in brief
(Lugtenberg and Kamilova, 2009).
Biological Nitrogen Fixation: Many associative and endophytic bacteria are now known to fix atmospheric nitrogen
and supply it to the associated host plants. A variety of nitrogen fixing bacteria like Arthrobacter, Azoarcus,
Azospirillum, Azotobacter, Bacillus, Beijerinckia, Derxia, Enterobacter, Gluconoacetobacter, Herbaspirillum,
Klebsiella, Pseudomonas, Serratia and Zoogloea have been isolated from the rhizosphere of various crops, which
contribute fixed nitrogen to the associated plants. For instance, contribution of 20 Kg N ha-1 by Azotobacter paspali
was demonstrated using 15N dilution technique (Baldani and Baldani, 2005; Reinhold-Hurek and Hurek, 2011). In
recent years, application of endophytic bacterial inoculants supplying N requirement efficiently to the various host

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plants including cereal crops have drawn attention for increasing plant yield in sustainable manner. Additionally,
some of the rhizobial isolates have also been found to colonize non-legume plant as an endophyte and benefit the
associating host (Rothballer et al., 2008). In terms of benefiting through nitrogen fixation, endophytic bacteria are
considered to be better than that of rhizospheric one as they provide fixed nitrogen directly to their host plant and fix
nitrogen more efficiently due to lower oxygen pressure in the interior of plants than that of soil.
When diazotrophic bacteria establishes endophytic association with plants, total content of plant nitrogen
rises which may be due to the biological nitrogen fixation or increased ability of nitrogen uptake from soil. In a well-
organized study in Brazil suggested that 60-80% of the accumulated nitrogen in different varieties of sugarcane
namely, CB45-3, SP70-1143 and Krakatau, was contributed by BNF (Boddey, 1995). Combination of nitrogen-fixing
bacteria (viz.,Rhizobium. trifolii and Burkholderia MG43) and reduced amount of chemical fertilizer can achieve
overall yield equivalent to the yield that was obtained from recommended full dose of chemical fertilizer
(Bhattacharjee et al., 2008). Gluconoacetobacter diazotrophicus is the main contributor of endophytic BNF in
sugarcane, which according to nitrogen balance studies fix as high as 150 Kg N ha-1yr-1 (Muthukumarasamy et al.,
2005). However, contribution of BNF to host may vary with the genotype of host. Proteomic analyses of sugarcane
variety SP70-1143 grown with G. diazotrophicus revealed up-regulated expression of ammonia lyase which indicates
increased metabolism resulted from increased uptake of nitrogen contributed by bacteria (Lery et al., 2011). Up-
regulation of genes for nitrogen metabolism during plant-bacteria interaction was also evident in differential gene
expression studies carried out earlier (Nogueira et al., 2001). Another nitrogen-fixing endophyte of considerable
interest is Azoarcus. This diazotroph inhabits the roots of kallar grass (Leptochloa fusca), which yields 20-40 t of hay
ha-1 yr-1 without the addition of any N fertilizer in saline sodic, alkaline soils having low fertility (Ladha and Reddy,
2000). Percent contribution of plant nitrogen as a result of BNF by few associating endophytic bacteria has been
given in table 1.
TABLE 1: Contribution of biological nitrogen fixation by associative/endophytic bacteria
*Nitrogen derived from air
At the molecular level, role of endophytic bacteria supplying fixed nitrogen to host was ascertained using non-
nitrogen fixing Klebsiella pneumoniae where the rice plants inoculated with non- nitrogen fixing K. pneumoniae in
nitrogen-deficient media showed signs of nitrogen deficiency on the contrary to the wild type counterpart (Iniguez et
al., 2004). Nitrogen-fixation ability of endophytic bacteria ex-planta or in-planta is measured or detected on the basis
of nif genes, encoding nitrogenase enzyme or by immunological detection of nitrogenase using antibody raised
against nitrogenase enzyme (Nogueira et al., 2001). Presence of structural genes namely nifH or nifD in associative
as well as endophytic bacteria have been detected by polymerase chain reaction using pair of universal primers (Jha
and Kumar, 2009; Reinhold-Hurek and Hurek, 2011). Expression of nif genes has also been demonstrated by
reverse transcription PCR (RT-PCR) from plants inoculated with Azoarcus BH72 and plants growing in field and in
other associative diazotrophic bacteria (Terakado-Tonooka et al., 2008; You et al., 2005).
Phosphate Solubilization: Phosphate is known to be the second most limiting compound for plant growth. Although
most of the soil is rich in phosphate but they are in insoluble form and cannot be utilized by plants or other soil
organisms. A vast number of PGPB with phosphate solubilizing property have been reported which include members
belonging to Burkholderia, Enterobacter, Pantoea, Pseudomonas, Citrobacter and Azotobacter (Park et al., 2010).
Some plant growth promoting bacteria solubilize phosphate from organic or inorganic bound phosphates and
Endophytic bacteria
Associa
ting
plant
%
Ndfa*
Reference
Rhizobium leguminosarum
bv.
trifolii
Rice 19 to 28 Yanni et al., 1997; Biswas
et al., 2000
Burkholderia
Rice 31 Baldani and Baldani, 2005
Herbaspirillum
Rice 19-47 Ladha and Reddy, 2000
Azospirillum
Rice 19-47 Ladha and Reddy, 2000
G. diazotrophicus, H. seropedicae, H.
rubrisubalbicans, A. amazonense and
Burkholderia sp
Sugarcane 29 Oliveira et al., 2002
K. pneumoniae
324
Rice 42 Iniguez et al., 2004
Burkholderia vietnamiensis
Rice 40-42 Govindrajan et al., 2008

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facilitate plant growth. Possible mechanisms for solubilization from organic bound phosphate involve either enzymes
namely C-P lyase, non- specific phosphatases and phytases. However, most of the bacterial genera solubilize
phosphate through the production of organic acids such as gluconate, ketogluconate, acetate, lactate, oxalate,
tartarate, succinate, citrate and glycolate (Khan et al., 2009). Type of organic acid produced for P solubilization may
depend upon the carbon source utilized as substrate. Highest P solubilization has been observed when glucose,
sucrose or galactose has been used as sole carbon source in the medium (Khan et al., 2009; Park et al., 2010).
Genetics and biochemical basis of acid secretion specifically gluconic acid in bacteria such as Erwinia herbicola,
Pseudomonas cepacia and Enterobacter asburiae have been reviewed by Rodriguez et al. (Rodrıguez et al., 2006).
Production of gluconic acid results from the conversion of glucose to gluconic acid by an enzyme glucose
dehydrogenase (Gcd). Gcd is a cell-envelope bound enzyme which depends on cofactor pyrroloquinoline quinine
(PQQ).
Production of Phytostimulating Compounds
PGPB exert its effects through the production of substances which stimulate plant growth. These substances include
phytohormones namely auxins, cytokinins, gibberellins, certain volatiles and the cofactor pyrroquinoline quinine
(PQQ). Several associative bacteria have been shown to produce auxins chiefly IAA, which enhances lateral root
growth formation and thus increase nutrient uptake by plants and root exudation, which in turn stimulates bacterial
colonization and thus amplify the inoculation effect. Plant growth promotion as a result of IAA has been documented
in several plants in recent years (Spaepen et al., 2007). However, beneficial effects of bacterial IAA depend upon the
optimum concentration, which may vary for different plants. The role of phytohormone produced by associative
bacteria in the promotion of plant growth during stress conditions such as salinity or draught has also been
demonstrated recently (Egamberdieva, 2009). Since, indigenously produced phytohormone in plants declines in salt
stress condition, salt tolerant associative bacteria may enhance plant growth by supplying phytohormones
synthesized by them. Similarly, IAA producing bacteria may enhance growth of plant in drought condition by
stimulating formation of well- developed root system enough for providing sufficient water from soil. Moreover, the
role of IAA in response to stress is evident from its increased production of IAA in Azospirillum sp. during carbon
limitation and acidic pH (Spaepen et al., 2007).
In addition to IAA, some of the associative bacteria have ability to produce other phytohormones such as
cytokinin and gibberellin. Cytokinin produced by Bacillus megatarium UMCV1, a rhizospheric bacterium, was found
to promote biomass production in Arabidopsis thaliana through the inhibition of primary root growth followed by
increased lateral root formation and root hair length of host plant (López-Bucio et al., 2007). Interestingly, few
isolates are capable of producing more than one phytohormone. Moreover, few bacteria namely B. subtilis, B.
amyloliqufaciens and E. cloacae promote plant growth through the production of volatile organic compounds (VOCs)
such as acetoin and 2,3-butanediol. VOCs of PGPR were found to enhance plant growth by regulating auxin
homeostasis in plants which was evident from induction of genes encoding enzymes of metabolism of IAA (Zhang et
al., 2008).
Induced Systemic Tolerance
A few PGPB enable the associating plants to tolerate abiotic stresses such as drought, salt, nutrient deficiency or
excess, extremes of temperature and, presence of toxic metals. Thus, physical and chemical changes in plants
resulted from PGPB-induced tolerance to abiotic stresses has been termed recently as ‘Induced Systemic Tolerance’
(IST). IST is elicited through the production of bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase,
antioxidants, cytokinin or VOCs (Fig. 2).

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Figure 2: Various components of induced systemic tolerance. PGPB may help associated plant reduce effect of
stressors through reducing level of stress ethylene due to presence of ACC deaminase activity, release of
antioxidants, volatile organic compounds and plant hormone cytokinin.
PGPB equipped with ability to synthesize ACC deaminase (ACCD) reduce level of stress ethylene produced in plants
in response to various biotic and abiotic stressors. ACCD degrades ACC, an immediate precursor of ethylene, to α-
ketobutyrate and ammonia (Yang et al., 2009). In addition to ACC deaminase mediated IST, other mechanisms also
exist to confer IST in response to stresses. In salt stress, level of Na+ elevates, which decreases plant growth and
productivity. The ion transporter high-affinity K+ transporter 1 (HKT1) regulates Na+ import in roots. VOC of Bacillus
subtilis GB03 confer salt tolerance by down- and up-regulating HKT1 in roots and shoots respectively, and result in
low Na+ accumulation throughout the plant in comparison to control. Other PGPB mediated IST include production of
cytokinin which affects abscicic acid (ABA) signaling of plants during stress and augmented production of antioxidant
catalase (Yang et al., 2009).
Rhizoremediation
Bacteria with the ability to degrade organic pollutant can be used for remediation of soil. Although pollutant degrading
bacteria characterized in laboratory environment may not thrive well in pollutant rich natural environment due to
requirement of energy for primary metabolism. Aforementioned problem can be overcome with the use of associative
and endophytic bacteria possessing ability to degrade soil pollutant. Since, PGPR colonizes in rhizosphere or
rhizoplane; they obtain their source of energy from root exudates for primary metabolism and degrade efficiently
organic xenobiotics present in the vicinity. For instance, P. putida PCL1444 effectively utilizes root exudates,
degrades naphthalene around the root, protects seeds from being killed by naphthalene, and allows the plant to grow
normally. Similarly, in-situ inoculation of P. putida W619-TCE reduced evapotranspiration of trichloroethylene by 90%
under field condition (de Bashan et al., 2012). In a recent report, endophytic bacteria isolated from seeds of Nicotiana
tabacum has been found to be potential candidate for reducing cadmium phytotoxicity (Mastretta et al., 2009).
Application of endophytic bacteria for degrading the pollutants like petroleum, toluene and other organic solvent as
well as protecting the plants from metals is of significant importance. In addition, endophytic bacteria engineered with
genes encoding enzymes for degradation of pollutants can be better exploited for remediation of soil (de Bashan et
al., 2012).

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Biocontroller
World agriculture faces a great loss every year incurred from infection by pathogenic organisms. Application of
microorganism for the control of diseases seems to be one of the most promising ways. Biocontrol systems are eco-
friendly, cost-efficient and involved in improving the soil consistency and maintenance of natural soil flora. To act
efficiently, the biocontrol agent should remain active under large range of conditions viz., varying pH, temperature
and concentrations of different ions. Biocontrol agents limit growth of pathogen as well as few nematodes and
insects. Biocontrol bacteria can limit pathogens directly by producing antagonistic substances, competition for iron,
detoxification or degradation of virulence factors; or indirectly by inducing Systemic Resistance (ISR) in plants
against certain diseases, signal interference, competition for nutrients and niches and interference with activity,
survival, germination and sporulation of the pathogen (Lugtenberg and Kamilova, 2009).
Antagonism: Associative/endophytic bacterial biocontrol agents may inhibit growth of fungal pathogens by one or
more of the several mechanisms, which include production of antibiotics, siderophore and lytic enzymes.
A vast array of antagonistic chemical compounds has been identified in bacterial biocontrol agents. Gram negative
biocontrol agents such as Pseudomonas produce HCN, pyoleutorin (PLT), pyrrolnitrin (PRN), 2,4-
diacetylphloroglucinol (2-DAPG) and phenazines (PHZ) chiefly phenazine-1-carboxylic acid and phenazine-1-
carboxamide Lugtenberg and Kamilova, 2009). The Role of each antibiotic produced by bacterial biocontrol agent in
conferring control of fungal pathogen may vary in different species. Control of Sclerotinia sclerotiorum by P.
chlororaphis PA23 is primarily executed by PRN while PHZ (phenazine-1-carboxylic acid, 2-hydroxyphenazine) helps
in the development of biofilm formation (Selin et al., 2010). On the contrary, PHZ (phenazine-1-carboxamide)
produced by P. chlororaphis strain 1391 was identified to be responsible for controlling tomato fruit and root rot
caused by Fusarium oxysporum f. sp. radicis-lycopersici. Few other biochemicals having pathogen inhibiting activity
include gluconic acid, 2-hexyl-5-propyl resorcinol, munumbicin, and few VOCs (2,3-butanediol) produced by
biocontrol agent (Backman and Sikora, 2008). The level of antibiotic synthesis depends upon nutritional factors viz.,
type of carbon source utilized, trace elements and availability of other nutrients as well as non-nutritional factors like
environmental influences. Regulation of antibiotic production in biocontrol bacterial agents involves GacA/GacR or
GrrA/GrrS, RpoD, and RpoS, N-acyl homoserine lactone (AHL) derivatives, and positive auto regulation (Compant et
al., 2005).
Under iron-limiting condition, some of the biocontrollers secrete siderophore, which chelates available iron of
the soil and sometime from cohabiting microorganism, and deprive pathogenic fungi from this element (Compant et
al., 2005). In addition to the role of siderophore in biocontrol, bacterial siderophore has been implicated in iron
nutrition of crop plants and heavy metal phytoextraction. Production of siderophore by diazotrophic bacteria seems
physiologically more important since the role of catecholate type of siderophore has been implicated in transport of
Mo under iron starved condition in Azospirillum lipoferum. Because nitrogen-fixing bacteria require both iron and Mo
for the activity of nitrogenase, the role of siderophore seems pivotal for any diazotrophic bacteria especially under
iron deficiency (Rajkumar et al., 2010).
Bacteria may limit growth of other microorganisms also through the production of hydrolytic enzymes such as
chitinase, β-1, 3-glucanase, protease and, laminarinase etc. For instance, Serratia marcescens and Paenibacillus sp.
secrete chitinase to exert antifungal activity against Sclerotium rolfsii and Fusarium oxysporum f. sp. cucumerinum
respectively. Bacillus cepacia destroys Rhizoctonia solani, R. rolfsii, and Pythium ultimum through the production of
β-1, 3-glucanase. Secretion of protease and chitinase was found to be the possible mechanism for antagonistic
activity of endophytic bacteria Enterobacter and Pantoea against fungal pathogen Fusarium oxysporum f.sp.
vasinfectum (Backman and Sikora, 2008; Compant et al., 2005).
Induced Systemic Resistance: Certain bacterial interactions with root enables the associated plant to develop
resistance against potent pathogens. This phenomenon is termed as Induced Systemic Resistance (ISR) and has
been noted to be exhibited by both associative and endophytic bacteria (Table 2) (van Loon, 2007). It was first
noticed in carnation and cucumber where inoculation with selected PGPB (rhizobacteria) reduced susceptibility to wilt
and foliar disease respectively. In contrast to many biocontrol mechanisms, extensive colonization of the root system
is not required for ISR to be exerted (Lugtenberg and Kamilova, 2009).

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TABLE 2: Biocontrol activity of associative/endophytic bacteria
The bacterial products that elicit induction of ISR are diverse and can induce in plants which possibly possess
receptor for respective ligands. These elicitors may be lipopolysaccharides, flagella, siderophores, antibiotics, VOCs
or quorum-sensing signals. Majority of ISR activated by PGPB is mediated by jasmonate or ethylene (van Loon,
2007). Mechanisms of ISR in Pseudomonas has been reviewed recently (Jankiewicz and Kołtonowicz, 2012). In a
recent study, plant growth promoting Bacillus cereus AR156 was found to trigger ISR in A. thaliana through SA- and
JA/ET-signaling pathways in an (Non-expressor of PR1) NPR1-dependent manner (Niu et al., 2011). Development of
ISR may induce various genes to strengthen the host plant mechanically or metabolically. It involves fortification of
plant cell wall strength, alteration of host physiology or metabolic responses and, enhanced synthesis of plant
defense chemicals such as phenolic compounds, pathogenicity related protein (PR-1, PR-2, PR-5), chitinases,
peroxidases, phenyl alanine ammonia lyase, phytoalexins, oxidase and/or chalcone synthase. These metabolic
products protect the host plant from future infections from pathogens. Local immune response induced by PGPR has
also been demonstrated in few studies. However, pattern of local immune response depends on genotype of plants
and respective bacterial species associated with them (Compant et al., 2005).
Biocontrol against Nematode: Few rhizobacteria acting as a biological control agent against plant-parasitic
nematodes have also been reported (Tian et al., 2007). Antagonistic activity by aerobic endospore-forming bacteria
(AEFB) (mainly Bacillus spp.) and Pseudomonas spp against nematodes is well known. It is mainly exerted by the
means of metabolic by-products, enzymes and toxins including 2, 4-DAPG (P. fluorescens), hydrogen sulphide,
chitinase, and hydrogen cyanide.
Colonization
Colonization of bacteria in rhizosphere or on plant surface is a complex process, which involve interplay between
several bacterial traits and genes. The colonization is multi-step process and includes (a) migration towards root
surface, (b) attachment, (c) distribution along root and (d) growth and survival of the population. For endophytic
bacteria one additional step is required that is entry into root and formation of microcolonies inter-or intracellularly.
Each trait may vary for different associative/endophytic bacteria. Colonization of bacteria is traced by tagging the
putative colonizing bacteria with a molecular marker such as auto fluorescent marker (e.g., green fluorescent protein
(gfp)) or β-glucosidase (gus) followed by microscopy (electron or confocal laser scanning microscopy) (Reinhold-
Hurek and Hurek, 2011). Fluorescent in-situ hybridization with real time PCR analysis can also be used for tracking
bacterial colonization and its quantification (Lacava et al., 2006). Understanding of molecular mechanism involved in
associative or endophytic colonization process is not well understood. Recent reports based on the genomic data
and other similar reports have suggested resemblance of colonization methods between pathogenic bacteria and
PGPB (Hardoim et al., 2008).
Endophytic Isolates
Plants
Path
ogenic Fungi/Bacteria
P. fluorescens
EP1
sugarcane
Colletotrichum falcatum
Burkholderia phytofirmans
PsJN
Grapevine
Botrytis cinerea
Burkholderia phytofirmans
PsJN
Tomato
Verticllium dahlia
P. Denitrificans
1
-
15
Oak
Ceratocystis fagacearum
P. puti
da
5
-
48
Oak
Ceratocystis fagacearum
P. fluorescens
63
-
28
tomato
F. oxysporum
f. sp.
radicis
-
lycopersici
P. fluorescens
63
-
28
pea
Pythium ultimum
and
F. oxysporum
f. sp.
pisi
Bacillus pumilus
SE34
Pea
F. oxysporum
f. sp.
Pisi
Bacillus pumilu
s
SE34
cotton
F. oxysporum
f. sp.
Vasinfectum
Bradyrhizobium
Sp. Strain ORS278
Arabidopsis
thaliana
transcriptome analysis based study
Paenibacillus alvei
K165
A. thaliana
Verticillium dahlia
Actinobacteria
A. thaliana
Quantitative PCR analysis based study
Bacillus cereus
AR156
A. thaliana
Pseudomonas syringae

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Root Colonization
Root colonization is the first and the critical step in establishment of plant-microbe association. Microorganisms move
towards rhizosphere in response to root exudates, which are rich in amino acids, organic acids, sugars, vitamins,
purines/ pyrimidines and other metabolic products. In addition to providing nutritional substances, plants start cross-
talk to microorganisms by secreting some signals which cause colonization by some bacteria while inhibits the other
(Bais et al., 2006; Compant et al., 2011). The patterns of chemoattractant especially organic acids may vary in
different isolates/strains. Malate, succinate and fructose are considered to be the strongest chemoattractants.
Exudate composition is in turn influenced by physiological status of plant, the presence of microbes and products
from rhizobacteria such as phenazines, 2,4-DAPG, zearalenone and exopolysaccharide. Sloughed up root cap cells
also have large impact on plant-microbe interaction. In addition to chemotaxis, electrotaxis (electrogenic ion transport
at the root surface) is also considered as a possible mechanism for initiating rhizobacterial colonization. Root hair
regions and emergence points are preferred site for colonization (Lugtenberg and Kamilova, 2009).
Colonization of root by microorganism may further induce release of exudates, and create ‘biased’
rhizosphere by exudating specific metabolic products. In some rhizospheric bacteria, root exudates induce flagellar
motility that leads their colonization on plant surfaces. During root colonization process, movement of associative
bacteria is followed by their adhesion on plant root which may be mediated by glycosylated polar flagellum, Role of
bacterial major outer membrane protein (MOMP) in early host recognition has been recognized in earlier report,
where MOMPs from Azospirillum brasilense showed stronger adhesion to extracts of cereals than extracts of
legumes and tomatoes. It suggests involvement of MOMPs in adhesion, root adsorption and cell aggregation of the
bacterium (Lugtenber and Kamilova, 2009). On the other hand, involvement of type IV pili and twitching motility has
been identified in tomato root colonization by Pseudomonas using pilA and pilT mutant, pilA is the gene encoding
prepilin, structural component of type IV pili and pilT encodes for protein required for pilus contraction that is
responsible for twitching motility (Lugtenberg and Kamilova, 2009; Reinhold-Hurek and Hurek, 2011). Preston et al.
(2001) identified SSIII secretion system III (SSIII) (hrp) in P. fluorescens SBW25 that is by in-vitro expression
technology (IVET), a promoter trapping technique. Moreover, role of two component regulatory system ColR/ColS in
competitive root colonization in P. fluorescence has been demonstrated. ColR/ColS system regulates
methyltransferase/WapQ operon, and thus maintains the integrity of outer membrane for efficient colonization (de
Weert et al., 2009).
Endophytic Colonization
Primary mechanism for colonization of endophytic bacteria is similar to that of associative one. Twitching motility and
type IV pile were found to be essential for successful colonization of Azoarcus, obligate endophytic bacteria (Böhm et
al., 2007; Reinhold-Hurek and Hurek, 2011). In addition, Bilal et al. (1993) suggested that cell-surface protein and
Ca2+ dependent twitching motility may be implicated in specific interaction with plants. Chemical composition of
lipopolysaccharides (LPS) present on the surface of bacteria might be determinative for successful colonization in
host plants (Serrato et al., 2010). Requirement for plant signal such as flavonoid present in root exudates of host
plant was also observed for stimulation of endophytic colonization of wheat and Brassica napus plants by
Azospirillum brsilense and A. caulinodans respectively (Lugtenberg and Kamilova, 2009).
Majority of natural isolates associated with plants form biofilm in the rhizosphere, on the surface of plant as
well as in the endorhizosphere. LapA (large adhesion protein A), a cell surface protein, or its homologue is supposed
to be putative adhesion needed for the adhesion of Pseudomonads on plant roots (Lugtenberg and Kamilova, 2009).
Entry of endophytic bacteria in plant roots is known to occur (a) through wounds particularly where lateral or
adventitious roots occur; (b) through root hairs and (c) between undamaged epidermal cells (Harodoim et al., 2008).
Chi et al. (2005) demonstrated that the colonization of gfp-tagged rhizobia in crop plants begin with surface
colonization of the rhizoplane at lateral root emergence, followed by endophytic colonization within roots, and then
ascending endophytic migration into the stem base, leaf sheath, and leaves where they develop high populations.
Azospirillum may also colonize endophytically through wounds and cracks of the plant root (Preito et al., 2011;
Reinhold-Hurek and Hurek, 2011).
Endophytic bacteria may colonize root tissues and spread actively in aerial parts of plants through
expressing moderate amount of degradative enzymes such as pectinases and cellulases. Utilization of aforesaid
enzymatic activities for colonization by Azospirillum irakense, Azoarcus sp. and others has been demonstrated as
one of the efficient methods to get entry into the host plant. Endoglucanase is one of the major determinants for the
colonization of endorhizosphere, which was evident from the observation that Azoarcus strain lacking endoglucanse
was not effective in colonizing the rice plants. The endoglucanase loosen larger cellulose fibers, which may help
entering to the plant. A homologue of endoglucanase gene has also been identified in P. stutzeri A1501, which

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occasionally colonizes cortex of crop plants. In addition to endoglucanse, exoglucanases may also help in
colonization process. An exoglucanase having cellobiohydrolase and β- glucosidase activity was identified to be key
player in colonization process of Azoarcus sp. BH72 (Reinhold-Hurek and Hurek, 2011). In Elaegnus and Mimosa,
the endophyte penetrates the radial walls presumably by digesting the middle lamella and then proceeds between
cells and through intercellular spaces. In contrast to above examples, genes encoding plant cell wall degrading
enzymes has not been found in endophytic bacteria Herbaspirillum seropedicae strain SmR1 (Pedrosa et al., 2011).
Azoarcus sp., an obligate endophyte of Kallar grass, has been critically studied by using transposon mutant
expressing β-glucuronidase (GUS) constitutively as a reporter gene (in Azoarcus sp. BH72). Azoarcus sp. BH72
colonize apical region of roots behind the meristem intensively and penetrate the rhizoplane preferentially in the zone
of elongation and differentiation. They colonize in the cortex region both inter- and intracellularly. In older parts of the
roots, they also occur in air spaces. Azoarcus sp. is capable of invading even the stele of rice and xylem vessels
suggesting systemic spreading into shoots through the transport in vessels (Hurek and Reinhold-Hurek, 2003). On
the contrary, shoot colonization of Gramineae appears to be more pronounced by G. diazotrophicus and H.
seropedicae (Jha et al., 2004). Furthermore, Compant and associates reported colonization of endophytic bacteria
Burkholderia phytofirmans in epidermis and xylem of even reproductive organ of grapevine. In another study Preito
and associates suggested that endophytic bacteria are confined within an organelle most likely vacuole which arises
by narrowing of an internal membranous structure in roots (Preito et al., 2011).
Endophytic colonization is not as specific as of Rhizobia but successful endophytic colonization does involve
a compatible host plant (Ryan et al., 2008). However, endophytic colonization indeed depends upon the physiological
changes in plants and is restricted or slowed down by defense mechanism (Rosenblueth and Martínez-Romero,
2006). Colonization of G. diazotrophicus was found to be diminished in plants grown under high nitrogen fertilizer
regime. This reduction in colonization was explained as a result of altered plant physiology in the presence of
nitrogen fertilizer, which reduces sucrose concentration to be utilized by endophytic bacteria. Influence of organic
amendment on endophytic population has also been demonstrated (Hallman et al., 1997). Plant defense response
plays critical role in regulating colonization of endophytic bacteria. In dicotyledonous plants, salicylic acid (SA) and
ethylene restricts endophytic colonization. Ethylene, a signal molecule of ISR in plants decreases endophytic
colonization as observed in Arabidopsis thaliana inoculated with K. pneumoniae 342 (Iniguez et al., 2005). However,
proteomic approach used to study colonization by bacteria indicated that jasmonic acid, not ethylene and SA,
contribute in restricting endophytic colonization in grasses (Miché et al., 2006). Expression of jasmoic acid (JA)
induced PR proteins (defense proteins) depends upon the compatibility of plant variety and endophytic bacteria.
Antimicrobial peptides synthesized by some plants like rice and maize may reduce endophytic colonization (Hurek
and Reinhold-Hurek, 2003). Understanding of molecular mechanism and conditions limiting the colonization process
need to be elucidated for exploiting the beneficial endophytic or associative interaction with plants.
Future Prospects and Challenges
A thorough exploration of associative/endophytic bacteria and their obvious abilities to enhance plant growth and
productivity indeed indicate the existence of natural associations of these bacteria and their beneficial impact which
can be exploited to feed burgeoning population of the world. Despite the fact that a large number of associative and
endophytic bacteria have shown plant growth promoting properties at laboratory and green house level, these
bacteria fail to exhibit consistent performance under natural conditions. The factors that affect colonization and thus
PGPB derived benefit to plant may be soil type, nutritional status of soil, host plant genotype and age as well as
climatic conditions (Bhattacharya and Jha, 2012). High amount of available utilizable nitrogen reduces colonization of
PGPB in natural condition and it may also reduce the process of nitrogen fixation due to regulatory mechanism acting
in the diazotrophic isolates. Therefore, a challenge is posed for systematic optimization for the application of suitable
PGPB isolates and the amount of fertilizer to be added to obtain maximum output. Use of compost may be useful at
some extent which provides utilizable nitrogen to support growth of microorganism and make the plant evade from
negative effects of PGPB colonization on it.
One of the major challenges includes selection of plant genotype and age, and compatible associative
bacteria. Understanding of this compatibility would help to enhance productivity by using specific strain for
inoculation. Since, the colonization of associative bacteria also depends upon seasonal changes and soil hydric
stress, multiples field trials are required to optimize parameters for obtaining the maximum output. Another factor
which is to be studied in details is the plant defense response which may limit or reduce the colonization of
associative bacteria. In addition, colonization mechanism is still not well understood. Intelligent analysis of genomic
and functional genomics studies can help manipulate the conditions to enhance colonization process and increased
plant growth properties.
Lastly and most importantly, extensive and intensive research on the understanding of associative and
endophytic ecology will be major determinant to maximize benefit from these bacteria.

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