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Endophytic Bacteria in Agricultural Crops

Article  in  Canadian Journal of Microbiology · February 2011

DOI: 10.1139/m97-131

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 Bacterial endophytes in agricultural crops
J. Hallmann, A. Quadt-Hallmann, W.F. Mahaffee, and J.W. Kloepper
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Oregon State University on 06/17/13

Abstract: Endophytic bacteria are ubiquitous in most plant species, residing latently or actively colonizing plant tissues
locally as well as systemically. Several definitions have been proposed for endophytic bacteria; in this review endophytes
will be defined as those bacteria that can be isolated from surface-disinfested plant tissue or extracted from within the
plant, and that do not visibly harm the plant. While this definition does not include nonextractable endophytic bacteria,
it is a practical definition based on experimental limitations and is inclusive of bacterial symbionts, as well as internal
plant-colonizing nonpathogenic bacteria with no known beneficial or detrimental effects on colonized plants. Historically,
endophytic bacteria have been thought to be weakly virulent plant pathogens but have recently been discovered to have
several beneficial effects on host plants, such as plant growth promotion and increased resistance against plant pathogens
and parasites. In general, endophytic bacteria originate from the epiphytic bacterial communities of the rhizosphere and
phylloplane, as well as from endophyte-infested seeds or planting materials. Besides gaining entrance to plants through
natural openings or wounds, endophytic bacteria appear to actively penetrate plant tissues using hydrolytic enzymes like
cellulase and pectinase. Since these enzymes are also produced by pathogens, more knowledge on their regulation and
expression is needed to distinguish endophytic bacteria from plant pathogens. In general, endophytic bacteria occur at
lower population densities than pathogens, and at least some of them do not induce a hypersensitive response in the
For personal use only.

plant, indicating that they are not recognized by the plant as pathogens. Evolutionarily, endophytes appear to be
intermediate between saprophytic bacteria and plant pathogens, but it can only be speculated as to whether they are
saprophytes evolving toward pathogens, or are more highly evolved than plant pathogens and conserve protective shelter
and nutrient supplies by not killing their host. Overall, the endophytic microfloral community is of dynamic structure
and is influenced by biotic and abiotic factors, with the plant itself constituting one of the major influencing factors.
Since endophytic bacteria rely on the nutritional supply offered by the plant, any parameter affecting the nutritional
status of the plant could consequently affect the endophytic community. This review summarizes part of the work being
done on endophytic bacteria, including their methodology, colonization, and establishment in the host plant, as well as
their role in plant-microbe interactions. In addition, speculative conclusions are raised on some points to stimulate
thought and research on endophytic bacteria.
Key words: endophytic bacteria, methods, localization, diversity, biological control.

Resume : Les bactCries endophytes sont omniprCsentes chez la plupart des espkces vCgCtales soit de faqon latente soit
colonisant activement, localement ou de faqon gCnCralisC les tissus des vCgCtaux. Plusieurs dCfinitions ont CtC propostes
pour les bactCries endophytes. Dans la prtsente revue, les endophytes vont Stre dCfinies comme ces bactCries qui peuvent
&tre isolCes de la surface de tissus vCgttaux dCsinfectCs, ou extraites de I'intCrieur des plantes mais sans dommage
visible pour ces dernibres. Bien que cette dCfinition n'englobe pas les bacttries endophytes non extractibles, c'est une
dkfinition pratique qui s'appuie sur les limitations expCrimentales et qui inclut les symbiotes bactkriens aussi bien que
les bactkries non pathogknes qui colonisent I'intCrieur des plantes, mais sans effet prkjudiciable ou bCnCfique pour les
plantes colonisees. Historiquement, les bacttries endophytes ont CtC considCrees comme des phytopathogbnes peu
virulents mais rCcemment, il leur a CtC reconnu plusieurs effets bCnCfiques pour les plantes-h6tes, en tant qu'agents qui
favorisent la croissance des plantes et augmentent la rCsistance aux phytopathogknes et autres parasites. En gCnCral, les
bactCries endophytes proviennent de communautCs bactCriennes Cpiphytes de rhizosphkres ou de phyllosphbres, aussi
bien que de graines ou de matCriels de propagation infest& d'endophytes. En plus de pCnCtrer dans les plantes par des
ouvertures naturelles ou par des blessures, les bactCries endophytes semblent utiliser activement des enzymes hydrolytiques,

Received March 26, 1997. Revision received July 14, 1997. Accepted July 15, 1997.
J. Hallmann,' A. Quadt-Hallmann,' W.F. Mahaffee,2 and J.W. K l ~ e p p e r Department
.~ of Plant Pathology, Biological Control
Institute, Alabama Agricultural Experiment Station, Auburn, AL 36849-5409, U.S.A.
' Present address: Institut f i r Pflanzenkrankheiten, Phytomedizin in Bodenokosystemen, NuBallee 9, D-53115 Bonn, Germany.
Present address: United States Department of Agriculture-Agriculture Research Service, Horticulture Crops Research
Laboratories, Biological Control Institute, Corvallis, OR 97330, U.S.A.
Author to whom all correspondence should be addressed (e-mail: jkloeppe@acesag.auburn.edu).
Can. J. Microbial. 43: 895 -914 (1997) O 1997 NRC Canada
 896 Can. J. Microbiol. Vol. 43, 1997

telles la cellulase et la pectinase, pour atteindre l'intkrieur des plantes. Du fait que ces enzymes soient aussi produites
par les phytopathogbnes, les connaissances sur leur rCgulation et leur expression devront Ctre accrues afin de distinguer
les bactkries endophytes des phytopathogknes. GCnCralement, les densitCs des populations des bactkries endophytes sont
moindres que celles des pathogbnes et, d'autre part, certaines d'entre elles n'induisent pas une rCponse d'hypersensibilitk
chez les plantes, ce qui indique qu'elles ne sont pas reconnues par leur h8te comme pathogbnes. Sur un plan kvolutif,
les endophytes semblent Ctre intermkdiaires entre les bactCries saprophytes et les phytopathogbnes, mais on ne saurait
que spkculer a savoir si les saprophytes kvoluent vers la pathogCnicitk ou si elles sont plus hautement Cvolukes que les
phytopathogbnes du fait qu'elles conservent un abri protecteur et une source d'C1Cments nutritifs en ne tuant pas leur
h8te. De surplus, la microflore endophyte est une structure dynamique influencke par des facteurs biotiques et
abiotiques; les plantes sont l'un des facteurs majeurs d'influence. Puisque les bactkries endophytes dependent de
nutriments offerts par les plantes, tout parambtre affectant le statut nutritionnel des plantes peut par conskquent affecter
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une communautk endophyte. Cette revue rtsume une partie des travaux dkja faits sur les bactkries endophytes, incluant
leur mkthodologie, la colonisation et 1'Ctablissement chez les plantes-hetes, ainsi que leur r8le dans les interactions
plante-microbe. De plus, des conclusions spCculatives sont avanckes sur certains points pour stimuler la penske et la
recherche sur les bactCries endophytes.
Mots elks : bactkries endophytes, mkthodes, localisation, diversitk, contr6le biologique.
[Traduit par la rkdaction]

Introduction the 1870s with work by Pasteur and others (reviewed by

Methods for examining bacterial endophytes Smith 1911; Hollis 1949). Since 1940 there have been
Isolation numerous reports on indigenous endophytic bacteria in vari-
Trituration and surface disinfestation ous plant tissues, including seeds and ovules (Mundt and
Vacuum and pressure extraction Hinkle 1976), tubers (Trevet and Hollis 1948), roots (Philip-
Centrifugation son and Blair 1957), stems and leaves (Henning and Villforth
For personal use only.

Recognition, localization, and enumeration 1940), and fruits (Samish et al. 1961; Sharrock et al. 1991).
Media Bacteria isolated from the internal plant tissue of healthy
Viable staining plants comprise over 129 species representing over 54 genera
Electron microscopy (Gardner et al. 1982; Hallmann et al. 1 9 9 7 ~ Mahaffee
; and
Immunological staining and quantification by Kloepper 1997; McInroy and Kloepper 1995a; Mundt and
ELISA Hinkel 1976; Sturz 1995), with Pseudomonas, Bacillus,
Nucleic acid hybridization Enterobacter, and Agrobacterium being the most commonly
Autoradiography isolated bacterial genera. Early reports regarded endophytic
Ecology bacteria as contaminants resulting from incomplete surface
Source of endophytic bacteria disinfestation or latent pathogens (Hollis 1949; Smith 191 1;
Mode of entry Thomas and Graham 1952). However, recent research has
Movement demonstrated that bacterial endophytes can improve plant
Localization growth and reduce disease symptoms caused by several plant
Population dynamics pathogens (Chen et al. 1995; Frommel et al. 1991; Kloepper
Indigenous endophytes et al. 1992b; Pleban et al. 1995; Van Peer and Schippers
Introduced endophytes 1989). Similarly, the low stress tolerance of axenic plants is
Membership and community structure commonly believed to result partly from the absence of
Influencing factors endophytic microorganisms. Recent research with endophytic
Biotic factors bacteria has primarily focused on these benefical effects
Plant-associated microorganisms (Chen et al. 1994; Frommel et al. 1991 ; Gardner et al. 1982;
Plant-parasitic nematodes Kempe and Sequeria 1983; Kloepper et al. 1992b; Lalande
Plant genotype et al. 1989; Scheffer 1983) and their ecology (Hallmann
Abiotic factors et al. 1 9 9 7 ~Mahaffee
; et al. 1997b; Mahaffee and Kloepper
Beneficial effects 1997; Misaghi and Donndelinger 1990; Musson 1994) to
Biological control of plant pathogens better understand how bacterial endophytes interact with
Plant growth promotion their hosts plants. The primary goal of this review is to dis-
Application cuss the theoretical and controversial aspects of bacterial
Future endophytes by summarizing at least part of the large but dis-
Acknowledgements persed literature published on endophytic bacteria since the
References 1920s.
Before beginning a discussion on bacterial endophytes,
Introduction the term endophyte must be defined. There are several defi-
The theory that nonpathogenic bacteria reside in plant tissues nitions of endophytes, which in general include fungi and
dates to Perotti (1926), but research on bacteria residing in bacteria (Chanway 1996). Fungal endophytes have been
the internal tissues of nonsymptomatic plants dates back to intensively reviewed in the last 10 years (Carroll 1988; Clay

@ 1997 NRC Canada

 Review I Synthese
1988; Siege1 et al. 1987), and this review focuses on the bac-
terial perspective. Conceptually, bacterial endophytes have
been defined by Kado (1992) as "bacteria that reside within
living plant tissues without doing substantive harm or gaining
benefit other than securing residency. " We consider this
definition to be too restrictive, in that it excludes the possibil-
ity that endophytes could, and probably do, form symbiotic
relationships with their host. Quispel (1992) considered
endophytes only as bacteria that establish an endosymbiosis
with the plant, whereby the plant receives an ecological
benefit from the presence of the symbiont, such as increased
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stress tolerance or plant growth promotion. Again, this defi-
nition is restrictive, in that only those bacteria that impart a
benefit to plants are considered endophytes, excluding those
that have a neutral or negligible effect on the plant. With
bacteria associated with the phylloplane, Beattie and Lindow
(1995) viewed the terms epiphytic and endophytic as two
ends of one spectrum reflecting the growth patterns of leaf
bacteria, rather than as two distinct g o u p s of organisms.
These authors suggest that an active exchange occurs between
the internal and external populations of one bacterial strain.
While this is an intriguing theory, there are most likely bac-
teria that are only capable of being either epiphytes or endo-
phytes.
- . To focus on plant-endophyte interactions in this
review, we consider any bacterium as an endophyte if it can
be isolated from surface-disinfested plant tissue or extracted
For personal use only.
from inside the plant, and if it does not visibly harm the
plant. This definition includes internal colonists with appar-
ently neutral behavior, as well as symbionts. It would also be
inclusive of bacteria that, during their endophytic phase,
fluctuate between endophytic and epiphytic colonization. We
feel more confident with this functional definition, since it
includes the broad spectrum of work being done on the
occurrence, population dynamics, and effect of nonpatho-
genic colonizers of internal plant tissue, and because it
reflects the experimental reality of sampling endophytes.
Methods for examining bacterial
endophytes
Methodology is extremely important and limiting in investi-
gations of bacterial endophytes. Because of their small size
and the laborious nature of most methods, knowledge of
bacterial endophytes is slow to progress. However, as more
and more researchers become involved with this group of
remarkable bacteria, we are beginning to realize the tremen-
dous potential for bacterial endophytes in agricultural pro-
duction systems.
Isolation
The isolation procedure for endophytic bacteria is of key
importance for research with endophytes and is, therefore,
part of the proposed definition of endophyte (see above).
Theoretically, any isolation procedure should recover the
complete internal population and only the internal popula-
tion; however, this is unlikely as a result of incomplete sur-
face disinfestation, the adsorption of bacterial cells to plant
cell structures, or penetration of the sterilant into plant tis-
sues resulting in the loss of some endophytes. Several tech-
niques have been employed for the isolation of endophytic
bacteria with each having advantages and disadvantages.
Researchers should select the specific technique that best
matches the objectives of the specific research question.
Tn'turation and surface disinfestation
The most common procedures for the isolation of endophytic
bacteria were based on trituration of surface-disinfested plant
tissue using various disinfectants, including sodium hypo-
chlorite (Fisher et al. 1992; Gardner et al. 1982; Hinton and
Bacon 1995; Quadt-Hallmann et al. 1997a, 1997b), ethanol
(Dong et al. 1994; Fisher et al. 1992; GagnC et al. 1987),
hydrogen peroxide (McInroy and Kloepper 1994; Misaghi
and Donndelinger 1990), mercuric chloride (Gagnt et al.
1987; Hollis 1951; Sriskandarajah et al. 1993), or a combi-
nation of two or more of these (Caetano-Anollts et al. 1990;
Pleban et al. 1995), followed by several washes in sterile
water or buffer solutions. Because of the high toxicity and
persistence of mercuric chloride, this disinfectant should be
restricted to plant tissues that cannot be disinfested by other
procedures. Surface detergents like Tween 20 (Mahaffee and
Kloepper 1997; Pleban et al. 1995), Tween 80 (Sturz 1995),
or Triton X-100 (Misaghi and Donndelinger 1990; Musson
et al. 1995) can be added to reduce surface tension of the
solvent, thereby allowing the disinfestant to reach protected
sites and grooves beyond collapsed epidermal cells on the
plant surface. Depending on plant species, age, and plant
parts (roots, stems, leaves, or seeds), concentrations of the
disinfectant being used vary and need to be optimized for
each situation. Another adaptation of the surface disinfestion
technique involves dipping plant tissue into ethanol and flam-
ing the surface (Dong et al. 1994).
Following surface disinfestation, the plant tissue is
triturated with a mortar and pestle or mechanical device
(e.g ., blender, KLECO tissue pulverizer (Mahaffee and
Kloepper 1997) in sterile water, buffer solutions, or liquid
media. The triturate is then processed for bacterial enumera-
tion (discussed below).
The trituration technique is dependent on complete sur-
face disinfestation, which is not always easy to achieve. In
theory, any contaminant on the surface should be killed or
rendered noncultureable without affecting any microorgan-
isms from within the plant tissue. Practically speaking, this
is not possible since disinfectants will also penetrate plant
tissue. The applied concentration necessary to kill the last
bacterial cells on the plant surface is most likely detrimental
to some bacteria inside the plant tissue, especially those in
the outer root cortex, thus leading to a biased sample and
reduction in total numbers of the indigenous endophytic com-
munity.
The extent of surface disinfestants penetrating into plant
tissues can be visually demonstrated by staining the root with
a tetrazolium-phosphate buffer solution (Patriquin and
Dobereiner 1978) and observing color development in bac-
terial cells. Since only actively metabolizing cells reduce
tetrazolium, bacterial cells killed by the surface disinfectant
would not have any color development. Another option
would be to use apoplastic dyes, such as disodium 4,4'-
bis(2-sulfostyryl) biphenyl or trisodium 3-hydroxy-5,8,10-
pyrenetrisulfonate (PTS), to monitor the relative progression
of the applied disinfectant (Peterson et al. 1981).
Because of these experimental limitations, one must often
compromise between an optimal surface disinfestation pro-
O 1997 NRC Canada

cedure and a procedure that reduces the penetration of the
surface disinfestant into the plant tissues. The extent of this
compromise will depend on the objectives of the research
and the amount of resources available (e.g., labor and plant
material).
To decrease the possibility of recovery of surface con-
taminants, sterility checks should be included to monitor the
efficiency of the disinfestation procedure for each sample.
Only if no bacterial growth occurs in the sterility check after
a certain time should the recovered bacteria be considered as
endophytes. The following sterility checks have been suc-
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cessfully applied: dipping roots in nutrient broth (GagnC
et al. 1987), transferring 0.1 mL of the final washing solu-
tion to a test tube with bacterial growth medium ( ~ c 1 n r o ~
and Kloepper 1994), or imprinting the surface-disinfested
plant tissue on nutrient medium (Pleban et al. 1995; Shishido
et al. 1995). A comparison of the above-mentioned three
methods resulted in similar contamination percentages for all
three techniques (Musson et al. 1995). Although these steril-
ity checks can help to detect insufficient surface disinfes-
tation, single bacterial cells or spores may still survive in
protected spaces under collapsed epidermal and cortical
cells, as demonstrated by ~ u a d t - ~ a l l m a nand
n Kloepper
(1996~)using electron microscopy.
The advantage of the trituration technique is that it can
be used for almost any plant tissue, including flowers, fruits,
For personal use only.
and seeds. Heterotrophic bacteria are recoverable from the
total internal plant tissue, and it is only minimally affected
by bacterial adsorption to plant parts, since the triturated tis-
sue is actually plated. However, this technique is not useful
for examining bacteria from certain physical niches (e.g.,
intercellular versus intracellular colonization. cortical versus
vascular tissue). In addition, sterility checks must be employed
for each sample; complete surface disinfestation is still diffi-
cult to assure, and hence, some surface colonizers might be
recovered as well. Although the KLECO tissue pulverizer
(Kinetic Laboratory Equipment Co., Visaila, Calif.) has
been effectively used for stem sections of cotton (Mahaffee
and Kloepper 1996) and cucumber (Mahaffee and Kloepper
1997) 2-3 cm in diameter, the trituration of woody tissue is
still problematic with most mechanical devices. In conclu-
sion.the trituration and surface disinfestation techniaues are
useful for describing the broad spectrum of endophytes
occurring in the total plant tissue.
Vacuum and pressure extraction
To bypass the above-mentioned problems concerning surface
disinfestation, alternative methods lacking any surface disin-
festation have been developed. A vacuum technique was
successfully used to extract xylem sap from the roots of
perennial plants like grapevine (Bell et al. 1995) and citrus
(Gardner et al. 1982), and the Scholander pressure bomb was
used for extracting root sap from cotton and other agricul-
tural crops (Hallmann et al. 1 9 9 7 ~ ) .Common to both
techniques is that the collected plant sap mainly consists of
fluid from the conducting elements and adjacent intercellular
spaces, representing two physical niches often considered
favorable for systemic colonizing bacteria.
A comparison of the bacterial community recovered from
cotton roots by the trituration and pressure bomb techniques
appears to indicate that only a subset of the bacterial
Can. J . Microbial. Vol. 43, 1997
endophytes are recovered by the pressure bomb technique.
Bacillus spp., shown to be a predominant group of the soil,
rhizosphere, and endorhiza communities of cucumber
(Mahaffee and Kloepper 1997), were only recovered by the
trituration technique, whereas Pseudomoms spp., commonly
reported as endophytes, dominated the bacterial community
recovered with the pressure bomb technique by Hallmann
et al. (1997~).Besides these qualitative differences, quanti-
tative differences in the total population size recovered by
these two approaches were also observed. The trituration
technique consistently gave higher numbers of endophytic
bacteria compared with either the pressure bomb (Hallmann
et al. 1997a) or vacuum extraction technique (Bell et al.
1995), which might be explained by the greater portion of the
physical niches in internal plant tissues being sampled com-
pared with the more selective isolation of vascular and inter-
cellular space colonists by the vacuum or pressure extraction
techniques.
Several controls, e.g., roots dipped in a bacterial suspen-
sion prior to extraction, should be included in experiments
using the pressure and vacuum extraction techniques to con-
firm that surface contaminants will not be forced from the
surface through the vascular system by the applied pressure
and vacuum, thus resulting in external tissue contaminants
appearing as endophytes (Gardner et al. 1982; Hallmann
et al. 1997~).To further eliminate the possibility of disrup-
tion of the plant cellular structure, the applied vacuum or
pressure should be released slowly and should not exceed
the permanent wilting point of the specific plant species.
~lthou~ theh pressure~andvacuum techniques likely estimate
lower total numbers of endophytic bacteria than does the
trituration procedure, they provide the benefit of selectively
isolating endophytes from the conductive elements which are
assumed to be systemic colonizers. The pressure and vacuum
techniques require robust plant material that can be held in
the required gasket and withstand the applied pressure or
vacuum. Soft tissues of herbaceous species or seedlings
might not withstand such treatment, thus limiting the practi-
cal use of these techniques to certain plant species or tissues.
Centrifugation
A third approach for recovery of endophytic bacteria from
plant tissue is centrifugation. This technique is commonly
used to collect the intercellular fluid of plant tissue (De Wit
and Spikrnan 1982), but would also extract endophytic bac-
teria from intercellular and vascular fluid similar to the
vacuum or pressure methods. Dong et al. (1994) used the
centrifugation technique to remove aseptic intercellular fluid
from sugarcane (Saccharum oflcinarum L.) stems. The
authors report that this fluid yielded pure cultures of an acid-
producing bacterium (approximately lo4 CFUImL) identical
to the type culture of Acetobacter diazotrophicus. Using the
centrifugation technique, the authors dipped stem sections of
3 -4 cm in length in ethanol and flamed the sections to kill
surface contaminants. Depending on the size of the sections,
they were centrifuged in Eppendorf tubes or larger glass
tubes. At centrifugation up to 3000 x g, most of the solution
of the apoplast could be removed. By applying cryo-scanning
electron microscopy (cryo-SEM) of the stem sections follow-
ing centrifugation, it could be shown that the cells were still
intact and no fluid from the symplast was displaced. How-
@ 1997 NRC Canada
 Review I Synthese
ever, this technique also required surface disinfestation of the
plant material, since the plant tissue will come into contact
with the extracted fluid during the centrifugation procedure.
Unlike the pressure and vacuum methods, the centrifugation
technique is suitable for soft plant tissue. Although the cen-
trifugation method requires time-consuming surface disin-
festation, several samples can be centrifuged at the same
time, though the overall time requirement might be less than
for the above mentioned techniques.
In summary, the decision as to which isolation method is
best for specific research objectives is influenced by criteria
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such as the physical niche of interest and plant-dependent
factors (species, age, and tissue), as well as available
resources and total number of samples being processed. The
application and comparison of different techniques offers the
advantage of examining endophytic colonization and locali-
zation from different perspectives, thus enhancing our under-
standing of the potential function endophytic bacteria have in
their hosts.
Recognition, localization, and enumeration
The isolation procedures for endophytic bacteria are simple
ways to demonstrate internal colonization and give a rough
estimate of the bacterial population density. However,
detailed studies on the occurrence of endophytic bacteria
within plant tissues require techniques that facilitate observa-
For personal use only.
tion of bacteria on a very small spatial scale and differen-
tiation of the bacterium of interest from the indigenous
microflora. The fact that some bacteria are attached to one
another or to the plant surface, thus bearing the risk of
underestimating total internal populations (Neyra et al.
1995), also needs to be considered.
In general, there are two types of investigations on the
population dynamics of bacterial endophytes: those that are
concerned with the indigenous populations and those that
monitor the colonization of an introduced endophyte. Both
types of investigations use similar methods and are generally
centered around plating techniques on selective or non-
selective media. However, immunological and nucleic acid
probes are evolving into powerful tools for examining bac-
terial populations.
Media
Probably the simplest technique for monitoring populations
of bacterial endophytes is their culturing on artificial media.
This technique is rapid, inexpensive, and sensitive. In general,
plant samples are processed, diluted, inoculated on assorted
microbiological media, and incubated under various environ-
mental conditions (Bell et al. 1995; Dobereiner et al. 1993;
Fisher et al 1992; GagnC et al. 1987; Jacobs et al. 1985;
McInroy and Kloepper 1 9 9 5 ~ Misaghi
; and Donndelinger
1990; Sturz 1995). The final population can then be esti-
mated by counting the colonies grown on the media after
considering the dilution factor or by applying the most proba-
ble number (MPN) technique (Sriskandarajah et al. 1993).
Several markers are available (reviewed by Kloepper and
Beauchamp 1992; Kluepfel 1993) to differentiate the intro-
duced microorganism from the indigenous microflora. A
common marker has been the selection of spontaneous
antibiotic-resistant derivatives of the wild-type strain, but
this approach might underestimate the actual population size
of the introduced strain (Kluepfel 1993; Mahaffee et al.
1 9 9 7 ~ ) Alternatively,
. heavy metal resistance or metabolic
markers can be used. Nevertheless, one must consider that
bacterial isolation based on artificial media only considers
viable microorganisms able to utilize nutrients of the selected
medium. The isolated microorganisms might represent only
a small fraction of the total endophytic population, a pheno-
menon commonly known for rhizosphere microorganisms
isolated on media (Kluepfel 1993).
Viable staining
Viable staining procedures overcome the problems associ-
ated with nonculturable microorganisms and can also be
applied for bacterial detection in situ. Therefore, vital stains
might provide a more realistic tool for enumeration and
localization of microbial communities. Bacterial conversion
of 2,3,5-triphenyltetrazoliumdichloride to red-colored fro-
mazans has been successfully applied to detect bacteria in
surface-disinfested corn roots (Patriquin and Dobereiner
1978). Based on an intense red color development, root areas
can be visually selected for further sectioning. However,
reduction of tetrazolium has also been considered as a mea-
sure of respiratory electron transport (Zimmermann et d .
1978), and plant particles of similar size to the bacteria, i.e.,
starch grains, might be stained too (Patriquin and Dobereiner
1978). Nevertheless, tetrazolium reduction by nontreated
plants usually takes more than 24 h and can, therefore, be
differentiated from bacteria-treated plants in which tetra-
zolium reduction occurs within 3 -4 h (Bashan and Holguin
1995). Alteratively, Bell et al. (1995) used acridine orange
to estimate the total number of bacteria extracted from grape-
vine xylem; however, the possible use of this technique for
in planta detection of bacteria was not assessed. In general,
viable staining procedures in combination with light micro-
scopy provide a rapid method to detect bacteria within plant
tissue and quantify total numbers of endophytic bacteria,
including nonculturable species in extracted plant sap.
Electron microscopy
Electron microscopy is a powerful tool to recognize and
localize endophytic bacteria in plant tissue. Conventional
scanning electron microscopy and cryo-SEM have been suc-
cessfully used to detect endophytic bacteria in various plants,
including corn (Turner et al. 1993), wheat (Gantar et al.
1991), fern (Van Doorn et al. 1991), and tomato (Sardi et al.
1992). Nevertheless, during the preparation of internal plant
tissues for SEM, the samples need to be cut, and thus bear
the risk of introducing bacterial contaminants from tissue
surfaces. Transmission electron microscopy reduces these
concerns, since the plant tissue with the bacterial endophytes
is first fixed and then sectioned. Within cross-sections, endo-
phytic bacteria can be distinguished from plant organelles
and plant-associated structures by a skilled electron micro-
scopist. This technique has been widely used to detect endo-
phytic bacteria in several plant species such as corn (Hinton
and Bacon 1995), wheat (Levanony et al. 1989; Ruppel et al.
1992), sugarcane (Dong et al. 1994), lupin (Wiehe et al.
1994), rice and Kallar grass (Hurek et al. 1994), potato
(Frommel et al. 1991), bean (Mahaffee et al. 1997a, 1997b),
cotton (Quadt-Hallmann and Kloepper 1996a), and pea
(Benhamou et al. 1996).
O 1997 NRC Canada

Overall, the small size of the bacteria, their heterogeneous
distribution in plant tissue, and the similarity in appearance
with cell organelles, as well as the low thickness of ultrathin
-
sections ( 60 nm), render endophytic bacteria difficult to
detect by conventional electron microscopy. Furthermore,
the detection limit for endophytic bacteria is considered to be
around 1 x lo5 CFUIcm root ( - 1 x lo6 CFUIg root)
(Wiehe et al. 1996), which is usually beyond average popula-
tion densities of 1 x lo4 - 1 x lo5 log CFUIg root.
Problems occurring during tissue preparation for microscopy,
such as access of fixatives or resins to uncut intercellular
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Oregon State University on 06/17/13
spaces due to heavily suberized and lignified parenchyma
cells or leakage of bacteria from the apoplast fluid of cut
spaces (Dong et al. 1994), increase the difficulties of detect-
ing endophytic bacteria within plant tissues. Nevertheless,
the resolving power of the electron microscopy make such
studies worth the effort. The newly introduced laser scanning
confocal microscopy technology also provides a promising
tool for in planta studies of endophytic bacteria (Sriskanda-
rajah et al. 1993).
Immunological staining and quant$cation by ELISA
Some of the difficulties associated with conventional light
and electron microscopy can be overcome by combining
microscopy with immunology. Immunological techniques
have been used for a long time to detect plant-associated
For personal use only.
microorganisms. Antibodies can be raised against proteinous
components of bacteria (e.g., fimbriae; Korhonen et al.
1986) or whole bacterial cells by injecting them into rabbits
or mice. For visualization, the antibodies themselves or
secondary antibodies raised against primary antibodies are
coupled with fluorochrome such as fluorescein isothio-
cyanate (FITC) (Mahaffee et al. 1997b; Schank et al. 1979;
Allan and Kelman 1977) or colloidal gold (Fig. 36) (Hurek
et al. 1994; Levanony et al. 1989; Quadt-Hallmann and
Kloepper 1996a; Sigee 1990). Immunofluorescence can then
be combined with light and electron microscopy, with pour
plate isolation (immunofluorescent colony staining (IFC))
(Mahaffee et al. 1997b; Van Vuurde and Van Henten 1983;
Van Vuurde and Roozen 1990) or with tissue printing
(Quadt-Hallmann and Kloepper 1 9 9 6 ~ )The. latter technique
involves sectioning of plant material and absorbing the cut
surface to a substrate (e.g., agar media, nitrocellulose
membranes) prior to antibody treatment. Tissue printing is
suitable for localizing bacterial endophytes in specific plant
tissues (e.g., cortical versus vascular tissue), but lacks the
resolving power to localize bacteria in intercellular spaces or
other detailed physical niches.
Depending on antibody sensitivity, single isolates or groups
of closely related microorganisms can be recognized using
immunological techniques. In some cases, antibody cross-
reactions are negligible (Levanony and Bashan 1989;
Levanonv et al. 1987, 1989: Schank et al. 1979). ,* whereas in
other cases, antibody specificity is limited and needs to be
characterized when analyzing samples (Quadt-Hallmann and
Kloepper 1996~).~ ~ ~ r o a c to h kenhance
s the specificity of
antibodies include the selection of specific bacterial cell wall
components as epitopes through extensive screening or the
adsorption of antiserum with cross-reactive antigens by
affinity chromatography (Hurek et al. 1994). Although
enhanced specificity of antibodies is usually desirable, it can
result in reduced sensitivity because fewer, but more
Can. J. Microbial. Vol. 43. 1997
specific, epitopes bind antibodies. This results in overall
lower levels of bound indicators, such as conjugate enzymes,
FITC , or coupled-gold particles. The sensitivity of immuno-
fluorescence techniques can be improved by amplifying the
antibody reaction (Van Vuurde 1991), increasing the effi-
ciency of the light emission by the bacterium, or modifying
the photographic system. By incorporation of the avidin-
biotin complex into either indirect or competitive enzyme-
linked immunosorbent assay (ELISA), quantitative detection
and enumeration of the bacteria can also be improved
(Levanony and Bashan 1990). Under optimal conditions the
visualization of single bacterial cells seems to be possible.
Nucleic acid hybridization
Endophytic bacteria can also be detected and identified
within plant tissue by in situ hybridization, a technique that
detects specific bacterial DNA or RNA sequences in plant
tissue. Nucleic acid probes are labeled by the attachment of
a hapten (i.e., biotin or digoxygenin), which is then recog-
nized by an antibody coupled to a visual marker, i.e., col-
loidal gold (McFadden 1991). Besides being useful for
bacterial localization, molecular techniques are sensitive
tools to detect endophytic bacteria in plant sap. Hurek et al.
(1994) used the polymerase chain reaction (PCR) to confirm
the presence of bacteria in the root and shoot tissues of rice
inoculated with Azoarcus sp. By selecting primers that
specifically amplify a characteristic DNA fragment of the
bacterial isolate, the PCR technique can distinguish between
subspecies of certain bacteria (De Boer and Ward 1995).
However, plant compounds can interfere with the detection
of bacterial endophytes by inhibiting the PCR. Precision can
be increased by exposure of the sample to microwaves to
promote cell lysis, followed by purification of the DNA by
ion-exchange chromatography (Eastwell et al. 1995).
Fusions of reporter genes such as lacZ (0-galactosidase)
with promoters derived from Azospirillum have also been
used to study internal root colonization (Katupitiya et al.
1995). Azospirillum bearing lacZ fusions were visualized by
staining plant tissue with the chromogenic @-galactosidase
enzyme substrate 5-brome-4-chloro-3-indolyl-0-galactoside
(X-gal).
The use of rRNA for the analysis of specific endophytic
microorganisms is another powerful tool because nucleic
acids possess highly conserved and unique regions (Kluepfel
1993). By combining rRNA hybridization with epifluores-
cence microscopy, even single cells are detectable (Amann
et al. 1990a, 1990b; Hahn et al. 1992). The main advantage
of all molecular techniques is their ability to detect both cul-
turable and nonculturable microorganisms.
Autoradiography
Autoradiography has also been used to study the dynamics of
endophytic bacteria in planta, since uptake of labeled pre-
cursor occurs only in molecules that are being actively syn-
thesized (Sigee 1990). Endophytic bacteria are labeled by
culturing on standard growth media containing (I5NH4)$O4
(You et al. 1995) or ['4C(U)]glucose (Pleban et al. 1995).
Following several washing steps to remove unincorporated
label, these bacteria can be inoculated into plants. The radio-
active labeled bacteria can finally be monitored using mass
spectrometry or autoradiography .
O 1997 NRC Canada
 Review I Synthese 901

Figs. 1-3. Electron transmission micrograph of cotton root cross sections with immunogold labeling of the bacterial endophyte
Pseudomonas jluorescens 89B-61. 89B-61 was applied as a seed treatment at 1 x 10' CFUIseed. Primary antibodies against 89B-61
were used at a dilution of 1:500 and secondary antibodies coupled with colloidal gold were used at a dilution of 1:40. Fig. 1. Bacterial
cells of 89B-61 located on the root surface and inside intercellular spaces close to the root epidermis. RS, root surface; EC, epidermal
cell. Bacteria are indicated by arrows. Scale bar = 10 pm. Fig. 2. Cross section of a cotton radicle after germination on water agar
and dipping in a suspension of JM22 (1 x lo7 CFUImL). Single cell of JM22 (arrow) located inside an intact root cortical cell (RC).
Scale bar = 10 pm. Fig. 3. Transmission electron microscopy of ultrathin sections of the root from a cotton plant seed-treated with
JM22 (1.0 x 109 CFUIseed). High numbers of JM22 cells are located in the intercellular spaces of the inner root cortex. (a) Root
cortex cell (RC). Scale bar = 10 pm. (b) Higher magnification of a ; gold label identifies bacteria as JM22. IS, intercellular space.
Scale bar = 1 pm. (Figure 1, from Quadt-Hallmann et al. 19976; Fig. 2, from Quadt-Hallmann et al. 1997a; Fig. 3, from Quadt-
Hallmann and Kloepper 1996. Reproduced with permission of Can. J. Microbial. O National Research Council of Canada.)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Oregon State University on 06/17/13
For personal use only.

Ecology tective environment for microorganisms than plant surfaces,

The capacity of bacteria to colonize the internal tissues of
plants could confer an ecological advantage over bacteria
that can only colonize plants epiphytically. The internal tis-
sues of plants are thought to provide a more uniform and pro-
where exposure to extreme environmental conditions such as
temperature, osmotic potentials, ultraviolet radiation, and
microbial competition are major factors limiting long-term
bacterial survival. However, there are probably other limit-
ing factors that must be overcome when establishing popula-
@ 1997 NRC Canada

tions in the internal tissues of plants. Thus, establishing and
maintaining an introduced bacterial population would still be
limited and influenced by the same factors that affect plant
health.
To further explore the potential use of endophytic bacteria,
a quantum leap in our understanding of how these bacteria
interact, colonize, and survive in plants is needed. The fol-
lowing are some of the ecological issues that have been
examined with speculation on what the results may mean and
ideas for future-research areas.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Oregon State University on 06/17/13
Source of endophytic bacteria
Two of the most frequently raised questions in connection
with endophytic bacteria are what is the origin of endophytes
and how do they enter plant tissues in nature? To answer the
first question, endophytic bacteria appear to originate from
seeds (Adams and Kloepper 1996; McInroy and Kloepper
1995b; Misaghi and Donndelinger 1990; Mundt and Hinkle
1976; Pleban et al. 1995), vegetative planting material (Dong
et al. 1994; Sturz 1995), rhizosphere soil (Hallmann et al.
1997c; Mahaffee and Kloepper 1997; Patriquin et al. 1983),
and the phylloplane (Beattie and Lindow 1995).
The importance of seeds as a source of endophytic bac-
teria is still controversial. Although endophytic bacteria have
been microscopically detected within seeds (Mukhopadhyay
et al. 1996), Mundt and Hinkle (1976) obtained endophytes
For personal use only.
only from damaged seeds. Since seed endophytes are mainly
associated with protected sites near the seed coat, they might
have colonized this area through fine openings in the seed
coat. This hypothesis is generated by the observed differ-
ences in the total number of endophytic bacteria isolated
from surface-disinfested seeds of cotton ranging from
1 x lo3 to 1 x lo5 CFUIg, while in sweet corn populations
were below 1 x. 10 CFUIg (McInroy and Kloepper 19956).
The authors explain these differences by referring to the seed
coat, which in the case of cotton is rough with deep grooves
and commonly damaged by the ginning process, thus repre-
senting a discontinuous protection barrier for the ovule. In
contrast, the seed coat of corn is smooth and normally intact,
providing a complete enclosure for the entire ovule. It still
remains possible that extremely low population densities of
endophytes are present in some seeds inside deeper regions
of the seed. This might explain the common observation that
endophyte populations increase from nondetectable levels in
seeds to detectable levels in radicles 48 h after placing
surface-disinfested seeds on water agar. Such bacterial endo-
phyte populations were either below detection limits or
in a physiological state that was nonculturable (Adams and
Kloepper 1996). Extensive cytological work is still necessary
to elucidate if endophytes are seed transmissible and to deter-
mine more closely the specific sites of colonization within
seeds.
The role of the spermosphere as a source of bacterial
endophytes is also evident by observations that bacterial
endophytes introduced as seed treatments colonized the inter-
nal tissues of root radicles newly emerged from the seed coat
(Mahaffee et al. 1997b; Musson et al. 1995). In detail,
endophytic colonization appeared to begin with migration of
bacteria through the germination slit and into the starchy
endosperm, from which these bacteria colonized the radicle
and coleoptile and finally spread systemically through the
plant (Hinton and Bacon 1995).
Can. J. Microbiol. Vol. 43, 1997
Besides seeds and spermosphere, several observations
favor the rhizosphere soil as the primary source for endo-
phytic colonization. Axenic potatoes planted into field soil
mainly harbored genera commonly found as soil saprophytes
(De Boer and Copeman 1974). Comparing the internal and
external bacterial communities of cucumber (Mahaffee and
Kloepper 1997), cotton (Hallmann et al. 1997c), and potato
(Sturz 1995), almost all endophytic bacteria were also found
in the rhizosphere, thus supporting the hypothesis that there
is a continuum of root-associated microorganisms from the
rhizosphere to rhizoplane to epidermis and cortex (Kloepper
et al. 1 9 9 2 ~ )Statistical
. analysis of bacterial diversity from
rhizosphere and endorhiza indicated that the initial compo-
sition of the endorhiza was dependent on the rhizosphere
community (Mahaffee and Kloepper 1997). However, the
endorhiza community quickly differentiated from that of
the rhizosphere with fewer genera present, suggesting that
the endorhiza (root interior) is a distinct habitat from the
rhizosphere (zone outside roots).
Mode of entry
With the exception of seed-transmitted bacteria, which are
already present in the plant, potential endophytes must first
colonize the root surface prior to entering the plant. Potential
internal colonists find their host by chemotaxis, electrotaxis,
or accidental encounter. Motility of beneficial associative
rhizosphere bacteria has been described for several bacteria
such as Alcaligenes faecalis, Azospirillum brasilense, and
Pseudomonasjluorescens (Bashan 1986a; You et al. 1995).
By using Tn5-induced mutants of Alcaligenes faecalis lack-
ing any response to attractants and mutants with a deficient
synthesis or overproduction of exopolysaccharides (EPSs),
attachment of Alcaligenes faecalis to rice roots could be
differentiated in three steps: bacterial adsorption onto the
rice root surface (rapid but weak interaction between bacteria
and host plant), anchoring on the root surface (strong inter-
action), and colonization of the root surface with some cells
entering into the rice root (You et al. 1995). A similar mode
of attachment is reported for Azospirillum brasilense to
wheat roots (Bashan and Holguin 1993; Michiels et al. 1991).
In general, entry into plant tissue can be via stomata,
lenticels, wounds (including broken trichomes), areas of
emergence of lateral roots, and germinating radicles (Huang
1986). However, the main entry for endophytic bacteria
appears to be through wounds that naturally occur as a result
of plant growth, or through root hairs and at epidermal con-
junctions (Sprent and de Faria 1988). Several authors have
reported extensive colonization of the secondary root emer-
gence zone (sites of root branches) by bacterial endophytes
(Agarwhal and Shende 1987; Jacobs et al. 1985; Lamb et al.
1996; Mahaffee et al. 1997b; Patriquin and Dobereiner
1978; Reinhold and Hurek 1988; Wiehe et al. 1994).
Because of breaks in the endodermis at these points, bacteria
colonizing the cortex can be observed to extend to and across
the endodermis into the vascular tissue (Mahaffee et al.
1997b; Patriquin and Dobereiner 1978; Reinhold and Hurek
1988). The importance of lateral root formation for bacterial
entry is underlined by the observation that Bacillus polymyxa
was recovered from inside pine seedlings only after lateral
roots had developed (Shishido et al. 1995). Plant wounding
in general, induced either by biotic (fungi, plant-parasitic
nematodes, insects) or abiotic factors (tillage, extreme tem-
@ 1997 NRC Canada
 Review I Synthese
perature fluctuations, grafting, root pruning) is ubiquitous in
any agroecosystem and is probably a major factor for bac-
terial entrance. For example, wounds artificially induced by
either adding nematodes to the soil (Hallmann et al. 1997b)
or carborundum to the bacterial inoculum suspension prior to
leaf inoculation (Quadt-Hallmann and Kloepper 1996a)
increased internal population densities of applied endophytic
bacteria. Besides providing entry avenues, wounds also
create favorable conditions for the approaching bacteria by
allowing leakage of plant exudates, which serve as a food
source for the bacteria.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Oregon State University on 06/17/13
Wounds and lateral roots are not, however, absolutely
required for entrance of endophytic bacteria. Seedlings grown
with minimal disturbance in liquid media or on water agar
were penetrated by endophytic bacteria long before lateral
root emergence (Levanony et al. 1989; Quadt-Hallmann
et al. 1997~).Since untreated control plants were endophyte
free, the observed bacterial behavior might indicate active
penetration. This hypothesis is supported by the presence of
cellulytic and pectinolytic enzymes produced by numerous
endophytic bacteria such as Azoarcus sp. (Hurek et al. 1994),
Azospirillum irakense (Khammas and Kaiser 199I), and
Pseudomonas jluorescens (Benhamou et al. 1996; Quadt-
Hallmann et al. 1997~).Enzymatic degradation of plant cell
walls by these bacteria was only observed when they colo-
nized the root epidermis but never after colonizing intercellu-
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lar spaces of the root cortex. These results suggest that the
endophyte induced production of cellulase and pectinase only
for penetration into the host plant. Although these observa-
tions demonstrate the possibility of active penetration mech-
anisms for some endophytic bacteria, very little is known
about the origin and regulation of these enzymes. Neverthe-
less, active penetration is still controversial and the fact that
soil bacteria show a higher frequency of hydrolytic enzymes
than xylem-inhabiting bacteria suggests that it is unlikely for
systemic endophytic bacteria to gain plant entry primarily via
production of hydrolytic enzymes (Bell et al. 1995). How-
ever, hydrolytic enzymes might only be produced by endo-
phytes during the early invasion phase and not after residing
in the plant tissue, as indicated by Benhamou et al. (1996).
In addition, one must differentiate between bacterial enzyme
production in vitro and in planta. Neither Enterobacter
asburiae JM22 nor Pseudomonas jluorescens 89B-61 pro-
duced cellulase when cultured on cellulose medium, although
cellulytic activity was observed in cross-sections of bacteria-
colonized cotton roots (Quadt-Hallmann and Kloepper 1996b).
However, constitutive release of plant cell wall degrading
enzymes by endophytic bacteria is undesirable since this
would confer plant pathogenicity (Collmer et al. 1982).
Therefore, endophytic bacteria must have some regulatory
mechanism to specifically regulate their enzyme production
a
in terms of quantity and time of expression, fact well
known for Xanthomonas campestris where virulent and
avirulent strains differ in their cellulase activity (Knosel and
Garber 1967).
Movement
Once inside plant tissue, endophytic bacteria either remain
localized in a specific plant tissue like the root cortex or
colonize the plant systemically by transport through the con-
ducting elements or apoplast (Hurek et al. 1994; James et al.
1994; Mahaffee et al. 1997b; Quadt-Hallmann et al. 1997b;
Patriquin and Dobereiner 1978). Bacterial movement has
been reported for intercellular spaces from 15 cells (Hurek
et al. 1994; Patriquin and Dobereiner 1978) to distances
representing 1-4 cm (Mahaffee et al. 19976). Systemic
spread of endophytic bacteria has been demonstrated for
Erwinia sp., which was recovered from cotton roots, stems,
and unopened flowers, as well as mesocarp and endocarp of
bolls (Misaghi and Donndelinger 1990), and for Pseudo-
monas aureofaciens, which has been shown to invade aerial
tissues of corn via internal movement from the roots (Lamb
et al. 1996).
The prevailing thought that systemic colonization of the
vascular system from cortical tissue is limited appears to be
due to the belief that the endodermis represents a physical
barrier to the apoplastic movement of bacterial endophytes
into the vascular tissue (Kloepper et al. 1992~).This theory
is based upon research demonstrating that the root endo-
dermis functions physiologically to regulate chemical flow to
the vascular region and that casparian strips, when intact,
limit apoplastic movement of solutes from the cortex to the
stele. Therefore, it was believed that endophytes present in
the root cortex could not traverse the endodermis to enter the
vascular system. However, as discussed above, the endoder-
mis is not an unbroken barrier since epidermal disrupture
occurs at the sites of secondary root formation, providing an
apoplastic route to the root stele, which can be visualized by
fluorescent dyes (Peterson et al. 1981). Bacterial endophytes
may also follow this same path to colonize the vascular tis-
sue. The endodermal barrier can also be surpassed by bac-
terial entrance via the undifferentiated cells of the root tip
(Mahaffee et al. 19976).
Nevertheless, systemic bacterial colonization of internal
plant tissues seems to be affected by the plant part. Although
both bacterial endophytes Pseudomonas jluorescens 89B-27
and Enterobacter asburiae JM22 colonized the cortical and
vascular root tissue of common bean, only JM22 colonized
the stem tissue following seed application (Mahaffee et al.
1997b). The authors postulated that limited spread of 89B-27
could be due to host recognition at the zone of differentiation
between root and stem tissue. A similar phenomenon was
observed by Quadt-Hallmann et al. (1997b) on cotton. While
JM22 colonized the total plant systemically, 89B-61 was
limited to endophytic colonization of the root cortex. Inter-
estingly, both 89B-27 and 89B-61 are known biological con-
trols agents (Liu et al. 1995a, 1995b, 1995c; Mahaffee et al.
1997a; Quadt-Hallmann et al. 1997a, 1997b), while for
JM22 no beneficial effects have been observed.
When considering systemic colonization of plants, the
question arises, is systemic colonization due to internal
movement by the bacterium or might bacteria be translocated
by capillary forces on the plant surface, from which they
reenter the plant tissue? For corn grown in a hydroponic
solution, inoculated Pseudomonas aureofaciens was found
on roots and seeds above the hydroponic solution, suggesting
capillary transport of Pseudomonas aureofaciens on the root
surface to the seeds but not further (Lamb et al. 1996). Since
the shoot surface was not contaminated with Pseudomonas
aureofaciens, any internal invasion of the aerial tissues
should have resulted from internal movement.
Once established within the plant tissue, endophytic bac-
teria can be transmitted vegetatively, such as reported for
Acetobacter diazotrophicus for two successive cuttings of
@ 1997 NRC Canada

sugarcane (Dong et al. 1994). The potential for seed
transmission of applied endophytes is still questionable, as
discussed before. However, endophytic bacteria are occa-
sionally isolated from surface-disinfested seeds, suggesting
that this question needs further research.
Localization
Bacterial preferences for colonization of specific plant areas
seem to be strain and species specific. In general, endophytic
bacteria colonize intercellular spaces (Figs. 1, 3a, and 3b)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Oregon State University on 06/17/13
(Dong et al. 1994; Hinton and Bacon 1995; Quadt-Hallmann
and Kloepper 1 9 9 6 ~ Mahaffee
; et al. 19976; Patriquin and
Dobereiner 1978; Reinhold and Hurek 1988; Ruppel et al.
1992; Wiehe et al. 1994), with only a few reports demonstrat-
ing intracellular colonization (Frommel et al. 199 1; Gantar
et al. 1991; Hurek et al. 1994; Jacobs et al. 1985; Mahaffee
et al. 19976; Quadt-Hallmann and Kloepper 1 9 9 6 ~ )Entero-
. the involvement of EPS in this phenomenon (Braun 1990).
bacter cloacea, for example, was observed in the intercellu-
lar spaces of corn roots, where it resided in the root cortex
and the stele just within the endodermis (Hinton and Bacon
1995). The diazotrophic endophyte Azoarcus sp. strain BH72
proliferated in the intercellular spaces of Kaller grass, moved
between cells of stele tissue, and finally entered parenchyma
cells (Hurek at al. 1994). The authors suggest that xylem
vessels and adjacent fibers might be colonized via penetra-
tion through pits. A similar colonization pattern was reported
For personal use only.
for Azospirillum spp. by Patriquin et al. (1983). However,
based on observations by Schank et al. (1979) and Levanony
et al. (1989) on grasses and wheat, Azospirillum brasilense
was only found in intercellular spaces of the root cortex and
never in the endodermal layer or the vascular system. Fur-
thermore, Azospirillum brasilense colonized the root tip,
elongation, and root-hair zone externally but was only
located within the cortex of the latter two zones (Levanony
et al. 1989). The lack of intercellular spaces in the interior
of the root tip and the meristematic zones seemed to be
responsible for the observed distribution pattern of Azospiril-
lum brasilense.
Besides residing in the intercellular space, some endophy-
tic bacteria also colonize plant cells intracellularly. While
this is not as common as intercellular colonization, intra-
cellular colonization might be functionally important. Jacobs
et al. (1985) observed nonpathogenic bacteria within paren-
chyma cells of sugar beet roots where the bacteria especially
occupied the large storage vacuoles of the cells. Bacteria
have also been detected intracellularly in grasses (Hurek
et al. 1994), wheat (Gantar et al. 1991), rice (You and Zhou
1989), bean (Mahaffee et al. 1997b), potato (Frommel et al.
1991), sugarcane (James et al. 1994), and cotton (Fig. 2)
(Quadt-Hallmann et al. 1 9 9 7 ~ )Confirmation
. of intracellular
colonization is often rendered by the thinness of the cross-
sections viewed, but these only represent a fraction of the
total plant cell. In the future, confocal laser microscopy
might overcome these limitations since this allows focusing
through entire intact plant cells.
Bacterial colonization of the vascular system has been
especially widely reported (Bell et al. 1995; GagnC et al.
1987; Gardner et al. 1982; Hurek et al. 1994; Lamb et al.
1996; Mahaffee et al. 19976; Ruppel et al. 1992; Samish
et al. 1961). However, it is still unknown if the vascular tis-
sue only serves as a transport channel for endophytic bacteria
Can. J. Microbiol. Vol. 43, 1997
or if bacteria actually multiply within the vascular system.
Considering the latter scenario, high numbers of bacteria
within the vessels might lead to plugging and, therefore,
could induce plant pathogenicity. This might explain why
endophytic bacteria are usually found in relatively low
numbers within the vascular system (Ruppel et al. 1992).
Similarly, Vasse et al. (1995) never observed nonpathogenic
isolates of endophytic Ralstonia solanacearum in the vascular
system, whereas pathogenic isolates colonized xylem vessels
consistently. For Ralstonia solanacearum it is known that
colonization of 25% of the vascular vessels is sufficient to
cause partial wilting of tomato plants (Vasse et al. 1995).
Furthermore, the slow growth and limited spread of an
EPS-deficient strain of Erwinia stewartii resulted in its con-
version from virulent to avirulent, indicating the importance
of bacterial growth and movement to induce symptoms and
These studies suggest that spatially limited colonization is
characteristic of endophytic bacteria and probably a major
factor in differentiating endophytic bacteria from plant
pathogens.
Although multiplication of endophytic bacteria in plant
tissue is difficult to demonstrate, results on long-term sur-
vival of several pseudomonads in citrus suggests a compati-
ble and dynamic association of the bacteria with the host
(Gardner et al. 1982). Hurek et al. (1994) reported inter- and
intra-cellular multiplication of Azoarcus sp. in the root cortex
of rice and Kaller grass as shown by electron microscopy. In
contrast, Pseudomonas aureofaciens did not remain viable in
stems of hydroponically grown corn, indicating an active
defense response of the plant, and the observed persistence
of Pseudomonas aureofaciens in soil-grown plants might be
due to continual invasion of the bacterium through newly
formed lateral and crown roots (Lamb et al. 1996). How-
ever, the fact that several endophytes approach and maintain
certain plateau population densities indicates bacterial multi-
plication. For example, Pseudomonas corruguta CC-471, intro-
duced at concentrations of 1.0 x lo3 and 1.0 x lo6 CFUI
mL into cotton stems, first increased within 3 days to
1.8 x lo6 and 4.3 x lo6 CFUIg stem tissue, respectively,
before the population finally decreased to 1.0 x lo3 CFUIg
at day 28, regardless of the inoculum densities (Chen et al.
1995). Similarly, bacterization of potato plantlets with
Pseudomonas sp. strain PsJN at 3.7 x lo5, 9.3 x lo6, and
2.8 x lo8 log CFUImL all resulted in a mean final root
population of 1.1 x lo5 - 1.0 x 106 CFUIcm root tissue
23 days later (Frommel et al. 1991). These results indicate
some optimum carrying capacity of endophytic populations
by the host plant, which fluctuates depending on plant age
and environmental factors. As with root colonization by
rhizobacteria, internal plant colonization by endophytes will
likely prove to be strain specific. Considerably more work is
needed to answer the question, do endophytes multiply inside
plants?
Bacterial endophyte growth and establishment within the
plant tissue requires nutrients, which leads to the question of
what type of nutrient source they utilize? Foster et al. (1983)
suggested that endophytes used exudates that enter the inter-
cellular spaces rather than break down the more complex cell
wall materials as the major food source. Cryo-analytical
SEM of corn roots indicated wet and dry intercellular spaces
O 1997 NRC Canada
 Review I Synthese
which both contained K and Ca, and occasionally small
amounts of S, P, and C1 (Canny and Huang 1993). Concen-
trations of the inorganic ions in the wet intercellular spaces
varied considerably from one intercellular space to the other,
which might explain the heterogeneous distribution of endo-
phytic bacteria often observed within plant tissue (Mahaffee
et al. 1997b; Quadt-Hallmann and Kloepper 1996~).The
distribution of ions is probably no artifact, because even
roots grown for 19 h in distilled water mist did not show any
change in concentration and the contents of the spaces did not
correlate with time of collection or distance from the root
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tip (Canny and Huang 1993). For sugarcane it has been
shown that the intercellular space is filled with fluid rich in
sucrose (12%), and that this fluid yielded pure cultures of an
endophytic bacterium identical with the type culture of Aceto-
bacter diazotrophicus (Dong et al. 1994). In general, research
regarding nutritional status of the apoplast and availability of
these nutrients for endophyte metabolism has been neglected
for a long time and should, therefore, be of increasing interest
in future studies on endophyte-plant interactions.
Population dynamics
An appropriate estimation of the total number of endophytic
.,
bacteria is often difficult as a result of the heterogeneous
distribution of bacteria within plant tissues and the fact that
some bacteria clump together in their secreted mucilage
For personal use only.
(Dong et al. 1994) or tend to adsorb to particles of any kind
including plant cell wall components (Fisher et al. 1992).
Evidence obtained by electron microscopy indicates that
most bacteria do not exist freely within xylem vessels, but
become entrapped in fibrous occlusions along border pits and
vessel walls (Gardner et al. 1982). This likely explains why
trituration of homogenized tissue results in 10- to 1000-fold
higher bacterial counts than vacuum or pressure extraction
(Gardner et al. 1982; Hallmann et al. 1997~).Additionally,
any enumeration technique based on bacterial cultivation on
solid nutrient media is &ought automatically to select for the
fraction of the total population that can grow on the chosen
medium under the chosen environmental conditions (Fisher
et al. 1992). Bell et al. (1995) compared bacterial popula-
tions estimated from vacuum-extracted xylem sap plated on
tryptic soy agar (TSA), direct staining with acridine orange,
and isolations of homogenized tissue of surface-disinfested
roots. Direct staining detected the highest number of endo-
phytic bacteria (6.10 x I@), followed by homogenized root
tissue (6.89 x lo3), and xylem sap plated on TSA (1.19 X
lo3). However, direct counting of acridine orange stained
cells by epifluorescence microscopy does not differentiate
between living and dead cells (Bell et al. 1995). Molecular
techniques also have their short comings, including ineffi-
cient lysis, inability to distinguish between living and dead or
inactive cells, and the absence of formation chimers that can
result in the detection of bacteria that do not exist. Again, the
choice of techniques will depend largely on the objectives of
the experiments and facilities and expertise available.
Indigenous endophytes
Population densities of bacterial endophytes seem to be
highest in the root and lower stem and decrease acropetally
(McInroy and Kloepper 1995b; Quadt-Hallmann and Kloepper
1996~).Common population sizes of indigenous endophytic
populations found in different crops ranged from 104 to
lo6 CFUIg fresh weight for cotton and sweet corn roots
and stems (McInroy and Kloepper 1994), 6.0 x lo3 to
4.3 x 104/gxylem of alfalfa (GagnC et al. 1987), 1.O X lo2
to 7.0 x 102/g xylem of citrus twigs, lo3 to lo6 CFUIg for
sugar beet roots (Jacobs et al. 1985), 0.4 x lo3 to 1.16 x
lo4 CFU/g cotton roots (Misaghi and Donndelinger 1990),
lo3 to 104 CFUIg roots for cotton (Hallmann et al. 1997a),
and to lo5 CFUIg root tissue of pine seedlings (Shishido
et al. 1995).
The total amount of endophytic bacteria that invade the
plant tissue is probably controlled by the plant and environ-
mental conditions, since not all potential endophytes (i.e.,
bacteria isolated from the internal tissues of plants) colonize
the internal tissues upon reintroduction (Musson 1994) and
both biotic and abiotic factors have strong influences on the
level of colonization (discussed below). For Alcaligenes
faecalis, an endophyte of rice, it has been estimated that only
10% of the bacterial population on the root surface entered
the root tissue (You and Zhou 1989).
Introduced endophytes
In general, average population densities of introduced endo-
phytic bacteria varied between 1 x lo3 and 1 x lo5 CFUIg
plant tissue for most investigated plants species (Dong et al.
1994; Frornmel et al. 1991; Quadt-Hallmann and Kloepper
1996a; Pleban et al. 1995). Similar to indigenous endo-
phytes, the highest bacterial densities are usually observed in
the roots and decrease from the stem to the leaves (Quadt-
Hallmann and Kloepper 1996a; Lamb et al. 1996). Overall,
population densities of endophytic bacteria are far below
threshold levels known for pathogenic bacteria, which can
range from 1 x lo7 CFUIg fresh weight, e.g., for Clavi-
bacter michigunensis subsp. sepedonicus on tomato (Tsiantos
and Stevens 1986), to 1 x lo9 - 1 x 101° CFUIg fresh
weight under severe disease pressure, as reported for Pseudo-
monas solanacearum on tomato, eggplant, and aubergine
(Grimault and Prior 1994). Nevertheless, single reports also
mention that endophytic bacteria can approach population
densities up to 1 x lo9 - 1 x 101° CFUIg fresh weight
under certain conditions without affecting plant growth
(Dimock et al. 1988; McInroy and Kloepper 1994). Inde-
pendent of the initial inoculum size, endophytic popula-
tions tend to approach optimal densities depending on the
plant tissue. For potatoes inoculated with Pseudomonas sp.,
the root population increased to a final population density of
1 x 106CFUIcm whereas the stem population decreased from
an initial concentration of 3.3 x lo5 - 1.9 x lo3 CFUIcm
(Frommel et al. 1991).
In terms of biocontrol activity, the importance of high
endophytic populations to guarantee plant benefits is uncer-
tain, because strain-specific factors, as well as different
modes of action (direct antagonism, release of plant growth
regulators, induced resistance), need to be considered.
Frommel et al. (1993) recorded a positive correlation of
potato growth stimulation and yield increase with the occur-
rence of high population densities of Pseudomonas sp. strain
PsJN on and in the potato roots. However, high populations
of Clavibacter xyli subsp. cynodontis, genetically modified
to express the delta-endotoxin of Bacillus thuringiensis in
corn, were associated with yield loss when the target pest
O 1997 NRC Canada

was absent (Tomasino et al. 1995). There is little question,
though, that an understanding of the population dynamics of
bacterial endophytes will enhance our ability to take advan-
tage of their beneficial characteristics.
Membership and community structure
Bacterial characterization by analysis of fatty acid methyl
esters (FAMEs) of total cellular fatty acids (Sasser 1990) has
replaced time-consuming identification methods based on
utilizing carbon sources for studies on bacterial ecology,
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since it allows processing of high numbers of bacteria in a
relatively short time. This technique is very useful in describ-
ing microbial communities. However, since the software
gives a similarity index of how the fatty acid spectrum of one
bacterium matches with the spectrum found in the library,
the number of bacterial entries in the library is of key impor-
tance. Using rarefraction analysis, Mahaffee and Kloepper
(1997) recommended sample sizes of at least 35 bacterial
isolates per sample for studies on bacterial community to
allow statistical processing of the data at the genus level. To
compare or characterize endophytic populations of different
plants or treatments, several diversity indices are available
(Ludwig and Reynolds 1988). Commonly used indices include
bacterial richness (number of species present), evenness (dis-
tribution of each species within a sample), or a combination
For personal use only.
of richness and evenness defined as Hill's diversity numbers
N 1 or N2, representing the number of very abundant species.
In general, Gram-negative species are found to be domi-
nant compared with Gram-positive strains, accounting for
78 % of the strains isolated from grapevine xylem (Bell et al.
1995), 84% from xylem of citrus roots (Gardner et al. 1982),
94% from alfalfa roots (GagnC et al. 1987), 75% from corn
and cotton tissue (McInroy and Kloepper 1994), and 100%
from cotton roots (Hallmann et al. 1997~).These results are
contradictory to Lalande et al. (1989), who found 88%
Gram-positive bacteria in 2-month-old roots of corn, and
Leifert et al. (1989), who reported an average of 70% Gram-
positive bacteria from nine micropropagated plant species
after 12 months.
Table 1 lists the most common taxa of endophytic bacteria
recovered from internal plant tissue. The main taxa belong
to the former Pseudomonas group (Pseudomonas, Burk-
holderia, Phyllobacterium) and Enterobacterceae (Entero-
bacter, Erwinia, Klebsiella) (Gardner et al. 1982; McInroy
and Kloepper 199%; Bell et al. 1995; Jacobs et al. 1985;
Hallmann et al. 1997~).
A comparison of the bacterial spectrum of a monocotyle-
donous (Zea mays L.) and a dicotyledonous (Gossypium
hirsutum L.) host yielded several endophytic species in cot-
ton that did not occur in corn and vice versa (McInroy and
Kloepper 1995a, 1995b). In addition, the bacterial spectrum
isolated from corn stems and roots was similar with only a
few exceptions, whereas for cotton, 14 taxonomic groups
present in roots were not found in stems.
Influencing factors
The colonization patterns of bacterial endophytes in plant
tissues is strongly dependent on numerous biotic and abiotic
factors that often interact. The effects of some of these fac-
tors have been investigated, as discussed below.
Can. J. Microbiol. Vol. 43, 1997
Biotic factors
Plant-associated microorganisms: Endophytic bacteria coexist
with other plant-colonizing microorganisms, such as viruses,
other bacteria, and fungi. Since space and nutrients provided
by the plant probably represent limiting factors for which
endophytic and pathogenic microbes must compete, several
interactive scenarios between microorganisms are imagina-
ble where competition, antibiosis, niche exclusion, symbio-
sis, and mutualism affect the attainable populations of a
particular bacterial endophyte.
Root infection with Rhizoctonia solani promoted coloni-
zation of two endophytes Enterobacter asburiae JM22 and
Pseudomonas fluorescens 89B-27, which extensively colo-
nized the necrotic tissue and extended 2 -4 cm into healthy
tissue (Mahaffee et al. 1997b). Analysis of the combined
incidence of bacterial and fungal endophytes in corn stems
revealed that areas of low fungal infection yielded the highest
bacterial counts (Fisher et al. 1992). In addition, endophytic
bacteria were more abundant in the root and lower stem
parts, whereas endophytic fungi dominated in the middle
stem. This antagonistic association of endophytic bacteria
and fungi in plant tissue might be of practical use for devel-
oping combinations of biological control agents covering
root and vascular pathogens.
Similar relationships have been observed for bacteria-
bacteria associations. Quadt-Hallmann et al. (19976) demon-
strated that coapplication of the systemic endophyte
Enterobacter asburiae JM22 with a rhizosphere colonist
(Arthrobacter agilis) did not affect internal population
density of JM22 when compared with a single application of
JM22. However, when JM22 was applied together with
another endophyte (Paenibacillus macerans), the coexistence
of both endophytes resulted in reduced population densities
of both endophytes compared with their single application.
The reduction in colonization was attributed to competition
for limited resources or physical space within the plant tis-
sue.
Very little is known about interactions between endophytic
bacteria and plant virus infection. For potatoes it has been
reported that potato virus X did not appear to affect the
endophytic bacterial flora in either stems or tubers (De Boer
and Copeman 1974). Direct antagonism between endophytic
bacteria and viruses seems to be negligible as a result of the
different habitats that they primarily colonize (bacteria, inter-
cellular space of roots and lower plant parts; virus, intracel-
lular in aerial plant parts). However, both organisms depend
on active plant metabolism and might compete for plant-
derived energy.
Plant-parasitic nematodes: The presence of plant-parasitic
nematodes was shown to increase total numbers of endo-
phytic bacteria (Hallmann et al. 1997b). Nematodes pene-
trate roots in the zone of elongation and differentiation
(Grundler and Wyss 1995), a region also preferably colo-
nized by saprophytic bacteria. Wounds created by the nema-
tode serve as avenues of entry for the bacteria, and in
addition, leakage of plant nutrients support a higher external
population of bacteria, thus representing a bigger potential
source of endophytic colonizers. Some bacteria might also be
attached to the juvenile cuticle and will be actively trans-
@ 1997 NRC Canada
 Review I Synthese 907

Table 1. Summary of indigenous endophytic bacterial genera most commonly isolated from various plant parts of different crops.

Plant part Bacterial taxaa Plant species Ref.

Seed Bacillus, Erwinia, Flavobacterium, Species (27), including cereal, Mundt and Hinkle 1976
Pseudomonas vegetables, and woody plants
Root Pseudomonas, Erwinia-like bacteria Alfalfa (Medicago sativa L.) GagnC et al. 1987
Root Bacillus, Pseudomonas, Corn (Zea mays L . ) Lalande et al. 1989
Corynebacterium
Root, radicle, stem,
unopened flowers,
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boll Erwinia, Bacillus, Clavibacter, Cotton (Gossypium hirsutum L.) Misaghi and Donndelinger 1990
Xanthomonas
Root Agrobacterium, Burkholderia, Cotton (Gossypium hirsutum L.) McInroy and Kloepper 1995a
Serratia
Root Burkholderia, Enterobacter Corn (Gossypium hirsutum L.) McInroy and Kloepper 1995a
Root Pseudomonas, Bacillus, Cucumber (Cucumis sativis L . ) Mahaffee and Kloepper 1997
Enterobacter, Agrobacterium,
Chryseobacterium, Burkholderia,
Arthrobacter, Stenotrophomonas
Root Pseudomonas, Enterobacter, Rough lemon (Citrus jambhiri Lush.) Gardner et al. 1982
Bacillus, Corynebacterium, and
other Gram-positive bacteria (e.g.,
Serratia)
Root Bacillus, Erwinia, Pseudomonas, Sugar beet (Beta vulgaris L.) Jacobs et al. 1985
Corynebacterium, Lactobacillus,
Xanthomonas
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Tuber Bacillus Potato Hollis 1951

Tuber Micrococcus, Pseudomonas, Potato (Solanum tuberosum L.) de Boer and Copeman 1974
Bacillus, Flavobacterium,
Xanthomonas, Agrobacterium,
coryneforms
Stem Enterobacter, Klebsiella, Corn (Zea mays L.) Fisher et al. 1992
Pseudomonas
Stem Bacillus Corn (Zea mays L.) McInroy and Kloepper 1995a
Stem Bacillus Cotton (Gossypium hirsutum L.) McInroy and Kloepper 1995a
Stem Enterobacter, Pseudomonas, Grapevine Bell et al. 1995
Pantoea, Rhodococcus
Fruit Pseudomonadaceae, Tomato Samish et al. 1961, 1963
Enterobacteriaceae, Cucumber
Achromobacteriaceae,
Micrococcaceae
aGenera are listed in order of reported abundance.

ported into the root tissue (Hallmann et al. 19976). Changes
in plant physiology following infestation of root-knot and
cyst nematodes might create favorable conditions for endo-
phytic bacteria as well. The endophytic bacterial spectrum
changes as early as 7 days following nematode infestation
(Hallmann et al. 19976).
Plant genotype: The broad use of cultivars with resistance
against bacterial pathogens raises the question, does host
genetics affect endophytic bacteria? Bell et al. (1995) com-
pared two grapevine cultivars differentiating in the degree of
resistance to crown gall to demonstrate that the plant geno-
type did not selectively affect endophytic bacteria within the
xylem. In contrast, differences in total endophytic popu-
lations are reported for tomato fruits of various cultivars
(Samish et al. 1961). It is also conceptionally possible that
pathogen resistance is not only due to the genetically con-
trolled plant characters but also to resistance conferred by an
altered endophytic community associated with plant resis-
tance, as reported for the multiadversity resistance cultivars
of cotton (Bird et al. 1980; Bird 1982).
Changes in plant physiology can lead to the development
of a distinct endophytic population (Hallmann et al. 1997c),
which raises the question of whether the plant itself controls
bacterial invasion by favoring some species and excluding
others. From the bacterial viewpoint, the transformation
from rhizosphere to endophytic colonization might be asso-
ciated with changes in the bacterial physiology. Van Peer
et al. (1990) found differences in several biochemical charac-
teristics between external and internal Pseudomonas species.
To the authors this indicated that bacterial penetration was
not just random and some form of selection occurred.

@ 1997 NRC Canada


However, it is possible that these biochemical differences
were a response to establishment in the new environment.
The complexity of various interactions between bacterial
endophytes and other microorganisms, parasites, and the
host plant are just beginning to be examined. An understand-
ing of these interactions will be the foundation on which the
successful application of these beneficial microorganisms
will be built.
Abiotic factors
The internal plant tissues provide a protective environment
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for endophytic bacteria. However, several factors (e.g., tem-
perature, rainfall, edaphic factors, UV radiation) that affect
the colonization of bacteria in the phylloplane and rhizo-
sphere will also likely influence the colonization and survival
of bacterial endophytes, though indirectly, since these
factors have a direct effect on the endophyte's host.
Nutrients appear to be more consistently available in
internal plant tissues, and competition among organisms may
be relatively low because of the presence of fewer types and
numbers of organisms. Nevertheless, the physical space for
colonization could be more limiting in the internal tissues of
plants. Soil physical and chemical factors, including pH,
salinity, and soil texture, are likely to affect endophytic
bacteria indirectly by altering the saprophytic bacterial com-
munity in the rhizosphere and, therefore, preselecting the
For personal use only.
source of potential endophytes. The type of planting sub-
strate may also play an important role in initial endophytic
colonization, since chemical composition and adsorption
capacity of the substrate can affect bacterial fitness and
motility (Quadt-Hallmann and Kloepper 1 9 9 6 ~ ) Following
. Enterobacter cloacae, an endophyte isolated from corn,
a drench with Enterobacter asburiae JM22, bacterial recov-
ery measured by ELISA was higher for siliceous sand, loamy
sand, or ground clay than for sandy loam and a peat-based
soil-less substrate (Quadt-Hallmann and Kloepper 1 9 9 6 ~ ) .
Mahaffee and Kloepper (1996) observed that colonization of
Pseudomonas jluorescens 89B-27 and JM22 in the common
bean was greater in sandy soils than in soils with low sand
content. This difference could be due to wounding of the root
as it grows through the coarse quartz crystals of sand. Under
field conditions. Samish et al. (1963) observed that for
different crops and different cultivars, endophytic bacteria
may appear abundantly in one field and rarely in others.
However, Bell et al. (1995) found no difference in endophy-
tic populations of grapevines grown at different vineyards.
Additionally, organic amendments have been shown to
influence endophytic populations. The incorporation of 1%
chitin to soil modified not only the bacterial spectrum in the
rhizosphere but also the endophytic community structure of
cotton roots (Hallmann et al. 1997~).Burkholderia cepacia
dominated the internal population (73%) of cotton roots
grown in chitin-amended soil but was rarely detected in
cotton grown in nonamended soil. Interestingly, Phyllobac-
terium rubiacearum, which showed slightly higher popu-
lation densities in chitin-amended soil, occurred only at low
numbers internally in cotton grown in chitin-amended soil
but was commonly (61 %) isolated from control plants. The
organic amendment may have altered the plant's physiology,
which would explain the observed shifts in the endophytic
bacterial community. The finding that organic amendments
are associated with modifications of endophytic communities
Can. J. Microbiol. Vol. 43, 1997
raises the possibility that targeted changes in crop endo-
phytes can contribute toward sustainable agriculture goals
such as biological disease control.
Beneficial effects
Biological control of plant pathogens
Endophytic bacteria colonize an ecological niche similar to
that of plant pathogens, especially vascular wilt pathogens,
which might favor them as candidates for biocontrol agents.
In addition, intensive work on rhizosphere biocontrol agents
has recentlv shown that five of six rhizobacteria, which
induced systemic resistance in cucumber, exhibitkd both
external and internal root colonization (Kloepper et al.
1992b). Exploiting an additional microbial habitat for
biocontrol purposes might enhance overall disease control
and increase control consistency, since the control agent
could avoid unfavorable conditions in one habitat by escap-
ing into the other habitat.
In targeting fungal diseases, endophytic bacteria have
shown significant control in model systems of Fusarium
oxysporu~f.sp. vasinfectum on cotton (Chen et al. 1995),
Verticillium albo-atrum and Rhizoctonia solani on potato
(Nowak et al. 1995), Rhizoctonia solani on cotton (Pleban
et al. 1995), and Sclerotium rolfsii on bean (Pleban et al.
1995). Similarly, several endophytic bacteria isolated from
rice seeds exhibited strong anti-fungal activity against Rhiz-
octonia solani, Pythium myriotylum, Gaeumannomyces
graminis, and Heterobasidium annosum (Mukhopadhyay
et al. 1996), and Hinton and Bacon (1995) reported that
exhibited in vitro antibiosis against Fusarium moniliforme.
Whereas the latter case indicates bacterial metabolites being
involved in the biological control of F. moniliforme, Chen
et al. (1994) suggested that the biocontrol affect of their
strains mainly resulted from enhanced host defense rather
than from bacterial metabolites. Duijff et al. (1997) refer to
the 0-antigenic side chain of the outer membrane lipopoly-
saccharides of Pseudomonas jluorescens WCS417r involved
in the induction of Fusarium wilt resistance in tomato.
Despite the numerous reports of greenhouse studies, only
limited field reports have confirmed that endophytes inocu-
lated onto plants confer biological control (Wei et al. 1996).
~ n t a ~ o n i s tactivity
ic of endophytic bacteria against bac-
terial pathogens has been reported for Clavibacter michigan-
ensis subsp. sepedonicum, the causal agent of bacterial ring
rot on potato (Van Buren et al. 1993). In addition, Pseudo-
monas jluorescens 89B-27 and Serratia marcescens 90-166
were observed to induce resistance in cucumber to Pseudo-
monas syringae pv. lachrymans, as well as to the fungal
pathogens Fusarium oxysporum f.sp. cucumerium and Col-
letrotrichurn orbiculare (Liu et al. 1995a, 1995b, 1 9 9 5 ~ ) .
Bacterial endophytes are also thought to play a role in the
resistance observed in the multiple adversity resistant (MAR)
cotton lines (Bird et al. 1980; Bird 1982).
There is some evidence that endophytes may contribute to
the control of plant-parasitic nematodes (Hallmann et al.
1995) and insects (Dimock et al. 1988). However, control of
these parasites seems to be more complex and difficult than
for fungal or bacterial pathogens, since damage from nema-
todes and insects occurs as a result of their feeding habit and
@ 1997 NRC Canada
 Review I Synthese
internal migration, thus limiting the efficacy of bacterial
antagonism. Nevertheless, sedentary plant-parasitic nema-
todes might be an interesting target for antagonistic endo-
phytes, since they stay localized within the plant for several
weeks and feed from a single feeding site.
A novel approach for the use of endophytic bacteria is the
manipulation of plant-associated microorganisms by molecu-
lar means (Dimock et al. 1988; Turner et al. 1993). Once
established, endophytic bacteria are strategically located for
delivering specific chemicals or signals manipulating plant
defense. Genetic engineering of selected endophytes offers
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several advantages: (i) interspecific gene movement, which
bypasses the limitations of traditional breeding; (ii) applying
the technology of engineering prokaryotes, which is still
more advanced than for eukaryotic organisms; (iii) engineer-
ing of bacteria, which may be more rapid than that required
to produce transgenic plants; (iv) potentially broad practical
use, since some endophytic bacteria can be applied to differ-
ent crop cultivars and species; and (v) possibility of addi-
tional bacterial applications after seeding depending on disease
pressure and plant development. Strategically, endophytes
should be viewed as potential vectors of foreign genes,
although considerably more research is necessary before the
practicality of this approach can be assessed.
Plant growth promotion
For personal use only.
Endophytic bacteria have been associated with the growth
promotion of several crops, including tomato, lettuce
(Bashan et al. 1989; Nowak et al. 1995; Van Peer and Schip-
pers 1989), potato (Frommel et al. 1991; Sturz 1995; Van
Peer and Schippers 1989), corn (Hinton and Bacon 1995;
Lalande et al. 1989), cucumber (Van Peer and Schippers
1989; Kloepper et al. 1992b; Nowak et al. 1995), rice
(Hurek et al. 1994), and cotton (Bashan et al. 1989). Accord-
ing to Sturz (1995), approximately 10% of bacterial isolates
recovered from within potato tubers were shown to promote
plant growth. The nonfluorescent Pseudomonas sp. strain
PsJN induced significant increases in potato root numbers
(24 - 196%), root dry weight (44 -201 %), and stem length
(26-28%), as well as enhanced leaf hair formation (55-
1lo%), secondary root branching, and total plant lignin con-
tent (43%) (Frommel et al. 1991).
Inoculation of rice with the diazotrophic endophyte
Azoarcus sp. strain BH72 significantly promoted plant
growth (Hurek et al. 1994). In this particular case, growth
promotion also occurred with Nif- mutants, indicating that
N2 fixation by Azoarcus sp. was apparently not involved in
plant growth promotion. Therefore, the authors speculated
that the observed plant growth promotion might have been
caused by enhanced plant mineral uptake and improved plant
water relationship associated with the colonization by
Azoarcus. Some strains of Pseudomonas, Enterobacter,
Staphylococcus, Azotobacter, and Azospirillum produce
plant growth regulators such as ethylene, auxins, or cyto-
kinins and have, therefore, been considered as casual agents
for altering plant growth and development (Arshad and
Frankenberger 1991; Bashan and Holguin 1997; Leifert
et al. 1994). In addition to a direct mechanism for growth
promotion, plant growth promotion is also thought to be due
to the suppression of a deleterious microflora by the intro-
duced endophyte (Kloepper et al. 1991). For example,
tomato seedlings inoculated with Pseudomonas sp. strain
WCS417r showed increased plant growth accompanied by
dense colonization of the applied strain in internal root
tissues and a consequent displacement of indigenous endo-
phytic pseudomonads that were deleterious to plant growth
(Van Peer and Schippers 1989).
The beneficial effects of bacterial endophytes vary but
appear to operate through similar mechanisms, as described
for plant growth promoting rhizobacteria (Kloepper et al.
1991; Hoflich et al. 1994). However, because of the differ-
ent habitats colonized, endophytes offer another tool for
developing biological control strategies. By combining the
use of bacterial endophytes with rhizosphere and foliar
antagonists, a holistic biological control system may be
developed that works against multiple pathogens of both root
and foliar tissue.
Application
To use endophytic bacteria in practical agronomic produc-
tion, reliable and practical methods of inoculum delivery
must be developed. In general, methods developed for appli-
cation of microbial inoculants in the rhizosphere and phyl-
losphere are also valid for endophytic bacteria (Andrews
1992; Baker and Henis 1990; McIntyre and Press 1991;
Stack et al. 1988). For the application of endophytic bacteria,
numerous methods have been used at the experimental level,
ranging from several variations of seed treatments and soil
drenches, to stem injections and foliar sprays of bacterial
suspensions (Fahey et al. 1991; Musson et al. 1995). Intense
testing of different delivery systems, including stab inocu-
lation into stems, soaking seeds in bacterial suspensions,
coating seeds with methyl cellulose, foliar spraying, vacuum
infiltration, and dipping pruned roots with different endo-
phytic bacteria indicated that the application method for
introducing endophytic bacteria into plant tissue is strain
specific (Musson et al. 1995). Crop Genetics International
Ltd. developed a seed inoculation technique by applying a
pressure differential to infuse the bacterial suspension into
imbibed seeds and redrying the seeds (Fahey et al. 1991;
Turner et al. 1993). The inoculated seeds met the requested
shelf-life requirements of several months, and application
of commonly used fungicides had no adverse impact on
bacterial survival or efficacy of the bacterial inoculation.
Several commercially oriented studies favor bacterial appli-
cation via alginate beads (Bashan 1986b). This has the advan-
tage of adding bacteria-specific nutrients like skim milk to
the alginate to improve bacterial survival rates. Bacterial
inoculation into plant cell suspensions and regenerated
embryos might be another possibility to guarantee early plant
colonization by the endophyte (Bashan and Holguin 1997).
Application of biocontrol agents to foliage and flowers
can be achieved by spraying bacterial suspensions or dust
formulations of lyophilized bacteria (Knudsen and Spurr
1987). However, little is known about optimal droplet sizes,
application pressures, and other technicalities (Sutton and
Peng 1993).
The advantage of seed treatment and soil drench is the
immediate protection of the seedling, while bacterial deliv-
ery via foliar sprays is an attractive method when applied in
combination with fungicides and insecticides. By combining
@ 1997 NRC Canada

different delivery techniques, endophytes can be applied
depending on need and growth stage of the crop of interest.
Overall, seed treatment with bacterial endophytes appears
to be an inexpensive, rapid, economically practical, and
reliable method for introducing endophytic bacteria. Never-
theless, the combination of seed treatment with soil drench
or leaf application may increase endophytic bacterial coloni-
zation within the plant and might enhance overall consistency
of beneficial effects. However, our knowledge of how
bacterial endophytes enter and colonize plants is limited and
development of successful application technologies will
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Oregon State University on 06/17/13
depend on improving our understanding of endophyte ecology.
Future
As our understanding of endophytic bacteria continues to
grow, the potential to capitalize on the unique characteristics
and close association with plants also grows. A variety of
possibilities are currently being explored, including the hard-
ening off and growth stimulation of axenic plants before
transplanting (Frommel et al. 1993; Nowak et al. 1995),
supply of additional nitrogen for the plant by diazotrophic
endophytes (Boddey 1995; Dobereiner et al. 1993; Hurek
et al. 1994), induction of systemic resistance against a broad
spectrum of pathogens (Kloepper et al. 1991; Liu et al.
1995a, 19956, 1995c), and the use of endophytes as delivery
For personal use only.
vehicles for crop protection agents, such as the toxin from
Bacillus thuringiensis (Dimock et al. 1988). There are proba-
bly many more uses for endophytic bacteria not yet envi-
sioned. In addition, plant benefits can also be enhanced by
the combined application of beneficial microorganisms, such
as endophytic colonizers of different ecological niches within
the plant (local and systemic colonizers, inter- and intra-
cellular colonization), the combined application of endophy-
tic bacteria with rhizosphere bacteria, or the combination of
bacterial and fungal endophytes.
However, to continue the development of endophytic bac-
teria as biocontrol agents and increase our ability to utilize
bacterial endophytes in agriculture, we still have to answer
the following questions. How do endophytes bypass plant
defense mechanisms and what food source do they utilize?
How are endophytic bacteria linked to plant resistance and
tolerance? Why and how do some bacterial endophytes colo-
nize the plant cells intracellularly? Could this character be an
evolutionary remnant of the endosymbiont theory for mito-
chondria and chloroplasts? Do these intracellular endophytes
communicate with the nucleus of the plant cell? If they do,
how can we take advantage of this communication link?
These are just some questions that may challenge our future
work in this area. The answers to these questions will decide
if endophytic bacteria are of potential use in practical agri-
culture.
Acknowledgements
We thank Jan Van Vuurde, Jan Van der Wolf, John
McInroy, Caroline Press, and George Musson for stimulat-
ing discussions that helped to form the concepts and ideas
discussed in this review. We also thank Johannes Kramer and
Vivian Vilich for critical review of this manuscript. This
work has been supported by research grants from the Alex-
ander von Humboldt Foundation, the German Research
Can. J. Microbiol. Vol. 43, 1997
Foundation, and the National Research Initiative of the
United States Department of Agriculture.
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