APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2002, p. 2198–2208 Vol. 68, No.

5
0099-2240/02/$04.00⫹0 DOI: 10.1128/AEM.68.5.2198–2208.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of Endophytic Colonizing Bacteria from

Agronomic Crops and Prairie Plants†
Denise K. Zinniel,1,2 Pat Lambrecht,1 N. Beth Harris,2 Zhengyu Feng,2 Daniel Kuczmarski,1‡
Phyllis Higley,1§ Carol A. Ishimaru,1储 Alahari Arunakumari,1# Raúl G. Barletta,2* and
Anne K. Vidaver1*
Departments of Plant Pathology1 and Veterinary and Biomedical Sciences,2 University of Nebraska, Lincoln, Nebraska 68583-0722
Received 6 September 2001/Accepted 4 February 2002

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Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endo-
phytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We
determined several phenotypic properties and found approximately equal numbers of gram-negative and
gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their
original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log10 CFU/g (fresh weight). More comprehensive
colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of
the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at
levels of 8.3 log10 CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes
were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log10 CFU/g (fresh
weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-
resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging be-
tween 3.4 and 6.1 log10 CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic
endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as
Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium isolates by 16S rRNA gene sequence, fatty acid,
and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbac-
terium testaceum. These microorganisms may be useful for biocontrol and other applications.

Endophytic bacteria are bacteria that live in plant tissues Endophytes inside a plant may either become localized at the
without doing substantive harm or gaining benefit other than point of entry or spread throughout the plant (17). These
residency (20, 21). Bacterial endophytes can be isolated from microorganisms can reside within cells (19), in the intercellular
surface-disinfected plant tissue or extracted from internal plant spaces, (31) or in the vascular system (3).
tissue (17). As cited in the extensive review of Kobayashi and Significant variations in the populations of both indige-
Palumbo (21), both gram-positive and gram-negative bacterial nous and introduced endophytes have been reported. These
endophytes have been isolated from several tissue types in variations are attributed to plant source, plant age, tissue
numerous plant species. Furthermore, several different bacte- type, time of sampling, and environment. Generally, bacte-
rial species have been isolated from a single plant (21). Endo- rial populations are larger in roots and decrease in the stems
phytes enter plant tissue primarily through the root zone; how- and leaves (24). Natural endophyte concentrations can vary
ever, aerial portions of plants, such as flowers, stems, and between 2.0 and 6.0 log10 CFU per g for alfalfa, sweet corn,
cotyledons, may also be used for entry (21). Specifically, the sugar beet, squash, cotton, and potato, as described by
bacteria enter tissues via germinating radicles (14), secondary Kobayashi and Palumbo (21). Similar results were obtained
roots (1), stomates (36), or as a result of foliar damage (25). for endophytic bacteria inoculated by root or seed drench-
ing, with the population levels reaching between 3.0 and 5.0
log10 CFU/g of plant tissue for tomato and potato (21). The
* Corresponding author. Mailing address for Anne K. Vidaver: De-
partment of Plant Pathology, University of Nebraska, 406C Plant Sci- levels of colonization by nonpathogenic endophytes tend to
ences Hall, Lincoln, NE 68583-0722. Phone: (402) 472-2858. Fax: (402) be far less than the levels of colonization by pathogenic
472-2853. E-mail: avidaver1@unl.edu. Mailing address for Raúl G. bacteria; the concentrations of the latter organisms range
Barletta: Department of Veterinary and Biomedical Sciences, Univer-
from 7.0 to 10.0 log10 CFU/g (fresh weight) of tissue in
sity of Nebraska, 211 Veterinary Basic Sciences, Lincoln, NE 68583-
0905. Phone: (402) 472-8543. Fax: (402) 472-9690. E-mail: susceptible infected plants (15, 45).
rbarletta@unl.edu. Our research goal was to determine the prevalence, proper-
† This is a contribution of the University of Nebraska Agricultural ties, persistence, and types of endophytic bacteria in agronomic
Research Division, Lincoln, Journal Series no. 13445.
and native plants. In this study, 853 different endophytic colo-
‡ Present address: Alex R. Masson Incorporated, Linwood, KS
66052. nizing bacterial strains were isolated from four agronomic crop
§ Present address: 7320 Raven Circle, Lincoln, NE 68506. species and 27 prairie plant species. Six of the most promising
储 Present address: Department of Bioagricultural Sciences and Pest colonizing strains were identified taxonomically as species of
Management, Colorado State University, Fort Collins, CO 80523-
Cellulomonas, Clavibacter, Curtobacterium, and Microbacte-
1177.
# Present address: Bristol-Myers Squibb Company, Pennington, NJ rium by fatty acid, carbon source utilization, and 16S rRNA
08534. gene sequence analyses.

2198
VOL. 68, 2002 ENDOPHYTIC BACTERIA IN PLANTS 2199

TABLE 1. Phenotypic characterization of endophytic bacteria from agronomic crops and prairie plants

No. of No. of Gram reactionc

Plant species (common name)
plantsa isolatesb,c Positive Negative NDd

Agronomic crops
Glycine max (soybean) 60 17 17e
Sorghum bicolor (sorghum) 120 353 (151) 137 (75) 173 (76) 43 (0)f
Triticum aestivum (wheat) 48 28 28 (0)e
Zea mays (corn) 90 336 (222) 164 (134) 136 (88) 36 (0)f
Total 318 734 (373) 301 (209) 309 (164) 124 (0)

Prairie plants
Agropyron elongatum (tall wheatgrass) 1 1 1
Agropyron intermedium (intermediate 1 1 1
wheatgrass)

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Amorpha canescens (leadplant) 5 4 1 3e
Andropogon gerardi (big bluestem) 6 7 3 4
Andropogon scoparius (little bluestem) 6 12 2 8 2e
Artemisia ludoviciana (cudweed sagewort) 3 2 1 1
Asclepias syriaca (milkweed) 28 20 20e
Asclepias verticillata (whorled milkweed) 4 2 1 1
Baptisia leucantha (white false indigo) 3 3 1 2
Bouteloua curtipendula (sideoats grama) 4 12 5 6 1e
Bouteloua gracilis (blue grama) 2 8 2 6
Bromus biebersteinii (meadow brome) 1 1 1
Bromus inermis (smooth brome) 1 1 1
Buchloe dactyloides (buffalograss) 2 6 1 4 1e
Callirhoe involucrata (purple poppy mallow) 2 3 2 1e
Dicanthelium oligosanthes (panicgrass) 6 3 3
Euphorbia podperae (leafy spurge) 3 2 1 1
Koeleria pyramidata (Junegrass) 4 3 2 1e
Lathyrus latifolius (perennial pea) 1 1 1
Lespedeza capitata (roundhead lespedeza) 2 2 2
Panicum virgatum (switchgrass) 7 7 2 4 1e
Petalostemon purpureus (purple prairieclover) 4 3 1 2e
Phalaris arundinacea (reed canarygrass) 1 1 1
Psoralea tenuiflora (wild alfalfa) 4 3 1 2e
Sorghastrum nutans (indiangrass) 7 7 2 4 1e
Sporobolus asper (prairie dropseed) 6 3 1 2
Vicia villosa (hairy vetch) 2 1 1
Total 116 119 25 59 35
a
Total number of individual plants surveyed.
b
Total number of bacterial isolates recovered from each plant species surveyed.
c
Number of isolates (number of corn and sorghum endophyte isolates utilized in more extensive phenotypic characterizations).
d
ND, not determined.
e
Gram reaction was not performed on the isolates due to culture loss in storage.
f
The Gram reaction test could not definitively determine the classification for the isolates.

MATERIALS AND METHODS (cultivars Mo17 ⫻ B73 and RN11) and sorghum (cultivars RS626 and Dekalb 61)
were randomly collected during the growing season (June to September) from 10
Bacterial strains. The type strain of Microbacterium testaceum, IFO 12675, was
healthy mature plants per site at four different geographical locations. Individual
received from Mariko Takeuchi, Institute for Fermentation, Osaka, Japan. A
total of 853 endophytic strains were isolated from diverse hosts. The following plants were severed aseptically 3 cm above the soil level, and the stalks were
strains were given special designations: CE648, isolated from dent corn; LB030, stripped of leaves, put into plastic bags, and kept on ice until further processing.
isolated from little bluestem; PD039, isolated from prairie dropseed; SE017 and In the laboratory, the stalks were wiped with 70% ethanol and flame sterilized,
SE034, isolated from sorghum; and SG041, isolated from sideoats grama. and each stalk was dissected into a segment containing the third, fourth, and fifth
Plant sources. A broad range of agronomic and prairie plants common to the nodes. A crosscut through the stalk 2 cm above the third node was made, and a
midwestern United States were surveyed for the presence of potential endo- sterile no. 8 cork borer was inserted to a depth of at least 2 cm. The outer stalk
phytic bacteria. Plants were selected based either on their economic importance was removed, exposing a cylinder of tissue inside the cork borer.
to agriculture or on their perennial nature and thus their potential ability to Soybean, wheat, and prairie plants were collected in the field during early
support stable bacterial ecosystems (Table 1). The agronomic plants screened summer (May and June) as described above. Plant leaves and stems were surface
were maize (corn), sorghum, soybeans, and wheat. The prairie plants tested sterilized for 10 s with 2% sodium hypochlorite containing 0.1% Tween 20
included various native species of grasses, forbs, legumes, and prairie wildflow- (Sigma-Aldrich Co., St. Louis, Mo.). To remove the disinfectant, sections were
ers. The agronomic plants were harvested from field plots located 300 miles apart rinsed five times each in two washes of nonsterile deionized distilled water and
in Nebraska, and the prairie plants were collected from three virgin prairies and a wash of sterile water; the sections were dried with sterile paper towels. All
an established prairie grass plot within 25 miles of Lincoln, Nebr. agronomic crop and prairie plant samples were placed into polyethylene bags
Isolation of endophytic bacteria. Putative endophytic bacterial strains were (Associated Bag Co., Milwaukee, Wis.) and either comminuted using a rolling
defined as isolates that were obtained from surface-sterilized plants, displayed press machine (Precision Machine Co., Lincoln, Nebr.) or dissected into ca. 1-cm
differentiable colony morphologies, and were recovered from the initial survey of pieces and macerated with either a sterile mortar and pestle or a sterile Polytron
agronomic crops and prairie grasses. For corn and sorghum, endophytic popu- homogenizer (Brinkman Instruments, Westbury, N.Y.). Tissue extracts were
lations were collected from the pith tissue of stalks. Samples of dent corn then serially diluted in 12.5 mM potassium phosphate buffer (pH 7.1) (phosphate
2200 ZINNIEL ET AL. APPL. ENVIRON. MICROBIOL.

buffer) and plated in triplicate to recover any bacterial endophytes present in the inoculation of seven replicates with 8.0 log10 CFU of bacteria/plant (homologous
plant tissue. studies) were conducted in the same manner, with minor modifications. Mono-
Preliminary studies were conducted to evaluate the efficacy of the decontam- cots lacking a dominant stem were inoculated in one to three stems per plant,
ination procedures. Stalks of corn and sorghum plants were sprayed with a and wheat crowns were injected with 100 ␮l of inoculum.
suspension containing 7.0 log10 CFU of the orange-pigmented organism Isolation of endophytic bacteria from experimentally inoculated plants. For
Clavibacter michiganensis subsp. nebraskensis per ml before the sterilization pro- the initial time point (1 day postinoculation), whole-plant samples of corn and
cedure described above was performed. Subsequent colony counting demon- sorghum were obtained as described above. For subsequent samples, partial
strated that the external bacterial recovery levels were 2.5 log10 CFU/g (fresh plant sections were collected from the lower main stem, the lowest nonsenescent
weight) or less. Without this procedure, virtually all the colonies recovered (6.0 leaf, a midstem leaf, and a newly expanded leaf. The only exception was for
to 7.0 log10 CFU/g [fresh weight]) were colonies of the sprayed bacteria. Thus, whole-plant samples of seedlings in the growth room studies. Plant sections were
our decontamination procedure effectively reduced potential external contami- surface sterilized as described above for soybean, wheat, and prairie plants.
nation by more than 10,000-fold. Before samples were pooled in polyethylene bags, a section that was 1 by 4 cm
Bacterial growth conditions. All bacteria were grown on plates at 27°C for 48 was cut aseptically from the interior stem tissue and a 5-cm-long sample was
to 72 h. Liquid cultures were grown for 16 h in a PsycroTherm shaking incubator dissected aseptically from the middle portion of the longitudinal axis of each leaf.
(New Brunswick Scientific, New Brunswick, N.J.) at 250 rpm. Bacto Agar (Difco Plant tissues were weighed, processed by the rolling press method, diluted, and
Laboratories, Detroit, Mich.) was added as a solidifying agent at a concentration plated as described above.

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of 15 g/liter when required. For initial isolation and phenotypic characterization, As a control for all inoculation studies to test for the presence of indigenous
the bacteria were grown on nutrient broth-yeast extract medium (NBY medium), endophytic bacteria, all agronomic crops and prairie plants were sham inoculated
Clavibacter michiganensis subsp. nebraskensis selective medium (CNS), or a mod- with phosphate buffer by using the methods used for the experimental plants. All
ified CNS medium without lithium chloride (16, 48). NBY broth and agar were bacterial endophytes recovered were compared qualitatively, which included
used to grow bacterial cultures for plant inoculation. Bacterial isolation in green- isolation on selective media and use of antibiotic resistance markers, and quan-
house studies and field trials was performed by using NBY agar containing 40 ␮g titative recovery data were compared with control data to distinguish growth of
of cycloheximide (Sigma-Aldrich Co.) per ml, modified CNS agar, and/or anti- introduced bacteria from the presence of indigenous microorganisms.
biotic-supplemented NBY agar. For genotypic characterization, bacteria were Growth room studies. Corn and sorghum endophytic bacterial strains were
grown in tryptic soy broth (Difco Laboratories) or on tryptic soy broth agar. tested in both aseptically grown cultivar Mo17 ⫻ B73 dent corn and cultivar
Phenotypic characterization of bacterial isolates. Colonies of bacterial isolates RS626 sorghum seedlings to screen for potential pathogenicity to host plants and
were characterized 48 and 96 h postinoculation for the following traits: color, to assess the ability of these bacteria to colonize agronomically significant crops.
form, elevation, margin, diameter, surface, opacity, and texture. Motility, mor- In conjunction with these assays, pathogenicity assays were also conducted in a
phology, size, and division mode were also evaluated by performing phase- greenhouse study. To prepare plants for in vitro inoculation, seeds were surface
contrast microscopy with a Zeiss universal microscope (Carl Zeiss, Inc., Thorn- sterilized by immersion for 20 min in a solution containing 0.80% sodium hypo-
wood, N.Y.) at a magnification of ⫻1,000 as described previously (8). The Gram chlorite and 0.05% Tween 20 with shaking at 250 rpm, followed by a 30-s dip in
reaction was performed as described previously (41) by using a 3% KOH test. 70% ethanol and two rinses in sterile distilled water. The seeds were then placed
Chitinase activity was assessed with shrimp chitin (Sigma-Aldrich Co.) by the embryo side up on NBY agar at 25°C until germination was observed in 2 to 4
method of Kole and Altosaar (22). Fatty acid analysis was performed by MIDI days. Seedlings were exposed to a photoperiod consisting of 16 h of light and 8 h
Laboratories using the Sherlock Microbial Identification System TSBA 3.0, of darkness. Seedlings that did not exhibit fungal or bacterial contamination and
TSBA 4.1, and CORYNE 1.2 software (MIDI, Newark, Del.), and carbon source had a coleoptile which was approximately 1 cm long were aseptically transferred
utilization was determined by using Biolog GP2 microplates comparing outputs to test tubes; each test tube contained a seed support chromatography paper wick
to the MicroLog System 2 database, release 4.01B (Biolog, Inc., Hayward, Calif.). (Whatman Inc., Clifton, N.J.) resting in Murashige and Skoog’s nutrient medium
Prototype strains used in taxonomic comparisons were obtained from the Deut- at pH 5.8 (Sigma-Aldrich Co.). Plants were harvested immediately after inocu-
sche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), the lation and at 1 and 8 days postinoculation.
Institute for Fermentation at Osaka (IFO), and the American Type Culture Homologous and host range greenhouse studies. All prairie plant species and
Collection (ATCC). wheat (cultivar Centurk) were evaluated for the ability to support colonization in
Antimicrobial agent resistance was tested individually on agar plates contain- the greenhouse of bacterial endophytes previously isolated from the same host.
ing antibiotics at the following concentrations: kanamycin, rifampin, streptomy- In addition, a host range study was conducted to determine the ability of corn
cin, and tetracycline, 10 ␮g/ml; and ampicillin, chloramphenicol, gentamicin, and and sorghum endophytes to colonize cultivars of cotton (Coker 404), cucumber
hygromycin, 50 ␮g/ml. Bacterial isolates were plated onto NBY agar with or (SMR-18), geranium (Aurora), milkweed (Natural Fiber Co.), onion (Spanish
without antibiotic supplementation. Most antibiotics were purchased from Sig- Yellow), potato (Red Kennebec), tobacco (Xanthi), tomato (Super Sioux), soy-
ma-Aldrich; the only exception was hygromycin, which was obtained from Roche bean (Amsoy 71), and wheat (Centurk). In both studies we utilized a randomized
Molecular Biochemicals, Indianapolis, Ind. Bacteria were considered sensitive to complete block design. To ensure that only replicating populations were recov-
an antibiotic at the concentration tested if no visible growth was observed on ered in both studies, the distal 15 cm of the main axis without the seed head was
plates containing the antibiotic when there was visible growth on control plates harvested from each plant 42 days postinoculation. Plants that were flowering or
after 48 h of incubation at 27°C. showing vegetative growth were preferentially selected.
Inoculation of plants with bacterial endophytes. Bacteria were grown to the Dent corn greenhouse studies and field trials. Wild-type bacterial strains from
mid-log phase, pelleted by centrifugation (3,440 ⫻ g, 10 min, 4°C), washed twice, corn and sorghum that colonized plant tissue at moderate to high concentrations
and suspended in phosphate buffer. To confirm inoculation density and purity, an without any apparent deleterious effects on seedlings grown in vitro were eval-
aliquot of each culture was serially diluted in phosphate buffer and plated on uated by using a randomized complete block experimental design for long-range
NBY medium. Corn and sorghum plants were inoculated in triplicate when a colonization (⬎50 days postinoculation). Cultivar Mo17 ⫻ B73 dent corn was
minimum height of 7.6 cm was reached and the stalks were at least 0.5 cm in used in greenhouse studies (University of Nebraska–Lincoln) and in two sepa-
diameter, which corresponded to 7 to 14 days after seed germination. A 26-gauge rate row-irrigated field trials located 32 miles apart at the University of Nebraska
needle attached to a tuberculin syringe containing a bacterial suspension was Agricultural Research and Development Center (Mead, Nebr.; field trial I) and
passed horizontally through the seedling stem approximately 1 cm above the the University of Nebraska Agronomy Farm (Lincoln, Nebr.; field trial II). In
crown of the plant. A 5-␮l droplet of suspension was formed at the tip of the addition, antibiotic-resistant mutants were included in these studies. Spontane-
needle, which was withdrawn through the plant stem. The plant was rotated 90°, ous antibiotic-resistant mutants were isolated from 14 bacterial endophytes by
the needle was reintroduced perpendicular to the first wound channel, and the the gradient plate method (29). Putative antibiotic-resistant mutants were con-
inoculation procedure was repeated. Thus, 10 ␮l corresponded to an inoculum of firmed by the same method and replicated onto NBY media containing 50 ␮g of
either ca. 6.0 log10 CFU/plant for growth room studies or 7.0 log10 CFU/plant for rifampin per ml, 25 ␮g of kanamycin per ml, or 12.5 ␮g of tetracycline per ml to
greenhouse studies and field trials. confirm resistance. Marker stability and growth rate (efficiency of plating) were
For soybean, wheat, host range, and prairie plant inoculations, bacterial sus- determined in vitro and in planta by isolation on NBY media and by three
pensions were prepared and quantified as described above. Plants were grown for consecutive transfers to NBY media without antibiotics and subsequent transfer
3 to 4 weeks until they reached the size described above. A 26-gauge needle was to NBY media supplemented with the appropriate antibiotics. After inoculation,
utilized for most inoculations; the only exception was the geranium inoculations, plants were harvested in the field after 24 h and at approximately 2-week inter-
for which the calibrated eye of a no. 20 tapestry needle was used. Inoculation of vals (five sampling periods) and in the greenhouse on day 1 and after about 2 and
nine replicates with ca. 6.0 log10 CFU of bacteria/plant (host range studies) and 7 weeks (three collection times).
VOL. 68, 2002 ENDOPHYTIC BACTERIA IN PLANTS 2201

Statistical analysis. The data were analyzed by using SAS, version 8 (SAS for the remaining isolates due to culture loss in storage or the
Institute Inc., Cary, N.C.), analysis of variance, as well as by using least-square variability of Gram reaction results.
means to test for pairwise differences when overall effects were present.
A more extensive phenotypic characterization was carried
16S rRNA gene amplification and sequencing. Genomic DNA was isolated by
using standard bacterial procedures (38). The following primers were used for out with 373 culturable microorganisms recovered from corn
PCR amplification of the 16S ribosomal DNA: p515FPL (5⬘-GTGCCAGCAG (222 isolates) and sorghum (151 isolates). A total of 209 of
CCGCGGTAA-3⬘) (35), p13B (5⬘-AGGCCCGGGAACGTATTCAC-3⬘) (34), these endophytes were confirmed to be gram-positive organ-
and PCR-1 (5⬘-AGTTTGATCCTGGCTCAGGA-3⬘). Each reaction mixture isms, and 164 were gram-negative organisms (Table 1). As
contained Taq DNA polymerase (Promega, Madison, Wis.), 1.5 mM magnesium
expected, more gram-negative bacteria than gram-positive bac-
chloride, each deoxynucleoside triphosphate at a concentration of 0.1 mM, 10%
(vol/vol) dimethyl sulfoxide (Fisher Scientific, Pittsburgh, Pa.), 0.4 mM spermi- teria were motile. Chitin utilization was positive for 157 gram-
dine (Sigma-Aldrich Co.), each primer (Integrated DNA Technologies, Cor- positive and 117 gram-negative isolates (data not shown).
alville, Iowa) at a concentration of 10 pM, and 10 ng of DNA per ␮l. The Analysis of resistance to various antibiotics showed that 20%
thermocycling conditions consisted of a denaturation step at 94°C for 3 min, 30 of the corn and sorghum isolates were sensitive to all of the
amplification cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 2 min, and
antibiotics at the concentrations tested (data not shown). For
a final polymerization step of 72°C for 4 min with a GeneAmp PCR System 9600

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(Perkin-Elmer, Norwalk, Conn.). PCR products were visualized on 0.8% agarose
gram-negative bacteria, resistance to ampicillin was most com-
gels, and the products were excised and purified either with glassmilk (GENE- mon (75%), followed by resistance to kanamycin (70%), resis-
CLEAN; Bio 101, Vista, Calif.) or with the Promega Wizard PCR Preps system tance to streptomycin (36%), and resistance to tetracycline
(Madison, Wis.) by following the manufacturers’ instructions prior to sequenc- (27%). Gram-positive strains demonstrated a similar pattern
ing.
of antibiotic resistance, although they were more likely to be
DNA sequencing was performed with an ABI 377 Prism DNA sequencer (PE
Applied Biosystems Inc., Foster City, Calif.). The following primers were used
resistant to kanamycin (51%) than to ampicillin (39%). Ninety
for sequencing: p91E (5⬘-TCAAA[G/T]GAATTGACGGGGGC-3⬘) (35), percent of both gram-positive and gram-negative strains were
SEQ-1 (5⬘-ACGTATTCACCGCAGCGTTG-3⬘), SEQ-2 (5⬘-GGCCTTCGGGT sensitive to rifampin.
TGTAAACCT-3⬘), and SEQ-3 (5⬘-CCAACATCTCACGACACGAG-3⬘). Also, Colonization studies with endophytic bacteria. To deter-
MIDI Laboratories sequenced 500 bp of the 5⬘ end using proprietary primers mine which of the endophytic bacteria had the ability to colo-
and procedures. Sequences were aligned, and a consensus sequence was com-
puted with fragment assembly tools in the Genetics Computer Group software
nize and persist at high levels in plant hosts, we carried out
package (Madison, Wis.). Nucleotide sequence similarities were determined by studies with experimentally inoculated plants. In all coloniza-
using BLAST, version 2.0 (National Center for Biotechnology Information da- tion studies, controls were included to verify that the inocu-
tabases). A phenogram was created by using PHYLIP software, version 3.5. lated bacteria were recovered. In general, the representative
Nucleotide sequence accession numbers. Partial sequence data for the 16S control plants inoculated with phosphate buffer yielded no
rRNA genes have been deposited in the EMBL/GenBank/DDBJ nucleotide
sequence data libraries. Data for endophtyic strains have been deposited under
indigenous bacteria. In addition, the colony morphologies of
the following accession numbers: CE648, AF474330; LB030, AF474326; PD039, the endophytes recovered after experimental inoculation were
AF474328; SE017, AF474325; SE034, AF474327; and SG041, AF474329. indistinguishable from the colony morphologies of the inocu-
lated organisms.
For the prairie endophytes, 86 isolates were inoculated into
RESULTS the homologous plant hosts and grown in the greenhouse. Of
these, 28 strains had colonized the plants at levels ranging from
Isolation and phenotypic characterization of bacterial en-
3.5 to 7.7 log10 CFU/g (fresh weight) at 42 days postinoculation
dophytes. To evaluate populations of potentially endophytic
(Table 2). The highest proportions of endophytes that were
bacteria in agronomic crops and prairie plants, a total of 853
able to colonize the original hosts in greenhouse studies were
isolates were collected over a 6-year period from all of the healthy
obtained from little bluestem, switchgrass, and prairie drop-
plants surveyed. For isolation of bacterial strains, plants were seed. Prairie endophytes LB030, PD039, and SG041 (see Ma-
harvested and processed as described in Materials and Methods. terials and Methods) were able to colonize the original hosts in
Following surface sterilization, wheat, soybean, and prairie plant a consistent manner at titers between 3.8 and 5.7 log10 CFU/g
samples were collected from the entire aerial portions of plants. (fresh weight) (data not shown). In the same homologous
The pith tissues of corn and sorghum stalks were chosen for greenhouse study, none of the 14 wheat endophytes tested was
isolation in order to assess potential relationships to the develop- able to colonize wheat to any significant extent.
ment of stalk rot disease, a common late season disease attributed A large number of endophytes were isolated from corn and
to multiple microorganisms (50). The population sizes depended sorghum, and we first performed preliminary growth room and
on the date of sampling and varied from 0 to 6.0 log10 CFU/tissue greenhouse assays to determine whether any of the isolates
sample (mean, 3.0 log10 CFU/tissue sample). A majority of the were pathogenic in dent corn and sorghum. The results indi-
microorganisms isolated (689 strains) were from corn and sor- cated that none of the 373 corn and sorghum isolates produced
ghum; 45 strains were recovered from soybean and wheat, and any disease symptoms or abnormalities in corn and sorghum.
119 strains were obtained from 27 different host species of Subsequently, to evaluate the colonization ability of corn and
grasses, forbs, legumes, and wildflowers (Table 1). As a whole, sorghum endophytes, all 373 isolates were tested with dent
fewer isolates were recovered from perennial plants than from the corn and sorghum seedlings which belong to the Andropogo-
agronomic crops. Preliminary characterization of these bacteria neae tribe. Of the 222 corn endophytes tested in corn seed-
showed that approximately equal percentages of gram-positive lings, 46 colonized corn seedlings at 8 days postinoculation at
(41%) and gram-negative (42%) bacteria were recovered from levels of 8.3 log10 CFU/plant or higher (Table 2). Similarly,
the agronomic crop plants, whereas gram-negative bacteria only 15 of the 151 sorghum endophytes tested in corn seedlings
(50%) were isolated more frequently than gram-positive bacteria colonized at these levels. In contrast, only 5 of 69 corn endo-
(21%) from prairie plants. Gram reactions were not determined phytes and 3 of 39 sorghum endophytes reached these coloni-
2202 ZINNIEL ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 2. Bacterial endophyte recovery from agronomic crops and prairie plants grown in either the greenhouse or the growth room
No. of isolates No. of isolates Colonization levelb
Plant host species
tested recovereda (avg ⫾ SD)

Greenhouse studiesc
Agronomic crop
Wheat 14 0
Prairie plants
Intermediate wheatgrass 1 0
Big bluestem 7 3 3.6 ⫾ 0.2 (3.5–3.9)
Little bluestem 10 6 4.7 ⫾ 1.3 (4.7 ⫾ 1.3 (3.6–7.0)
Cudweed sagewort 2 1 4.5
Milkweed 15 0
Whorled milkweed 2 0
Sideoats grama 11 3 5.0 ⫾ 0.9 (4.0–5.7)
Blue grama 8 3 5.3 ⫾ 1.5 (3.6–6.4)

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Meadow brome 1 0
Smooth brome 1 0
Buffalograss 5 2 5.2 ⫾ 0.0 (5.2–5.2)
Leafy spurge 2 0
Perennial pea 1 0
Roundhead lespedeza 2 0
Switchgrass 6 5 4.6 ⫾ 0.9 (4.0–5.5)
Purple prairieclover 1 0
Reed canarygrass 1 1 3.9
Indiangrass 6 2 6.2 ⫾ 2.1 (4.8–7.7)
Prairie dropseed 3 2 5.5 ⫾ 0.1 (5.5–5.6)
Hairy vetch 1 0
Total 100 28

Growth room studiesd

Agronomic crops
Corn 217e 207 (95) 7.1 ⫾ 1.4 (3.2–9.4)
145f 134 (92) 6.8 ⫾ 1.5 (3.0–9.6)
Sorghum 69g 67 (97) 6.5 ⫾ 1.3 (3.1–8.7)
39h 37 (95) 6.7 ⫾ 1.6 (3.1–8.7)
Total 470 445 (95)
a
Number of endophytic bacterial strains that were confirmed to colonize plant tissue. The numbers in parentheses are percentages.
b
Titer for growth chamber studies at 8 days postinoculation expressed as log10 CFU/plant and titer for greenhouse studies at 42 days postinoculation expressed as
log10 CFU/g (fresh weight). The values in parentheses are the lowest and highest average titers.
c
All bacterial isolates were reintroduced into the same plant species at a level of 8.0 log10 CFU/plant.
d
All bacterial isolates were reintroduced at a level of 6.0 log10 CFU/plant.
e
Number of corn endophytes that were reinoculated into corn seedlings.
f
Number of sorghum endophytes that were reinoculated into corn seedlings.
g
Number of corn endophytes that were reinoculated into sorghum seedlings.
h
Number of sorghum endophytes that were reinoculated into sorghum seedlings.

zation levels in sorghum seedlings; five of these isolates colo- colonize corn seedlings at titers ranging from 9.0 to 9.6 log10
nized both corn and sorghum seedlings. Of the endophytes CFU/plant (data not shown). Likewise, all of these isolates
inoculated into corn and sorghum seedlings, six strains ap- colonized sorghum seedlings at levels of 6.6 to 8.6 log10 CFU/
peared to be specific for sorghum and one strain appeared to plant. These isolates also exhibited colonization levels of 0.8 to
be specific for corn. In summary, 69 strains were recovered 4.7 log10 CFU/g (fresh weight) over an extended host range.
from either corn or sorghum seedlings at desirable concentra- All three colonized geranium and cucumber; in addition,
tions (8.3 log10 CFU/plant or higher). SE017 colonized wheat, potato, and tomato, while SE034 grew
Based on the growth room assays described above, we se- in tomato and milkweed (data not shown).
lected 19 corn endophytes and 10 sorghum endophytes to Extensive long-range greenhouse studies and field trials
perform host range studies in the greenhouse in order to test were performed with nine bacterial endophytes from corn
the ability of these microorganisms to colonize diverse plant and eight bacterial endophytes from sorghum showing sig-
species, including those listed in Table 3. Cucumber and to- nificant colonization levels in at least one of the types of
mato plants supported growth of about 60% of the corn and seedlings. These strains were introduced into greenhouse-
sorghum endophytes, respectively. Most endophytes (26 of 29 grown dent corn in order to determine the titers achievable
strains) were able to colonize at least one species different throughout most of the corn plant life cycle, through day 67
from the original host at levels ranging from 0.1 to 5.8 log10 postinoculation (Table 4). All strains were recovered at the
CFU/g (fresh weight), and two endophytes were able to colo- highest levels early in the study (days 1 and 11), and the
nize five or six plant species. average colonization levels were 6.1 ⫾ 0.1 log10 CFU/g
The corn and sorghum endophytes CE648, SE017, and (fresh weight). By day 67, the colonization levels had de-
SE034 (see Materials and Methods) were able to consistently creased by about 10-fold (Table 4). The colonization levels
VOL. 68, 2002 ENDOPHYTIC BACTERIA IN PLANTS 2203

TABLE 3. Corn and sorghum endophyte populations in a range of hosts determined in greenhouse studies
Corn endophytes Sorghum endophytes
Plant host species
b
(common name) No. of Colonization level No. of Colonization levelb
isolatesa (avg ⫾ SD) isolatesa (avg ⫾ SD)

Allium cepa (onion) 17 (8) 3.9 ⫾ 1.2 (2.4–5.8) 7 (2) 3.5 ⫾ 0.8 (2.9–4.1)
Asclepias syriaca (milkweed) 19 (0) 0 10 (2) 1.3 ⫾ 0.4 (1.0–1.5)
Cucumis sativus (cucumber) 19 (11) 1.5 ⫾ 1.2 (0.1–3.8) 10 (5) 1.3 ⫾ 1.3 (0.4–3.5)
Pelargonium ⫻ hortorum (geranium) 19 (2) 1.9 ⫾ 0.1 (1.8–1.9) 10 (4) 3.2 ⫾ 1.3 (1.4–4.3)
Glycine max (soybean) NDc ND 7 (0) 0
Lycopersicon esculentum (tomato) 19 (4) 1.7 ⫾ 1.0 (1.0–3.1) 10 (6) 2.0 ⫾ 1.6 (0.8–4.6)
Nicotiana tabacum (tobacco) 16 (4) 3.0 ⫾ 1.0 (2.3–4.5) 10 (3) 2.7 ⫾ 0.2 (2.5–2.8)
Solanum tuberosum (potato) 19 (6) 2.8 ⫾ 0.3 (2.5–3.2) 10 (5) 3.0 ⫾ 0.4 (2.7–3.6)
Triticum aestivum (wheat) 15 (2) 3.1 ⫾ 1.5 (2.0–4.1) 5 (2) 4.6 ⫾ 0.1 (4.5–4.7)
a
Number of isolates tested (inoculated at a level of 6.0 log10 CFU/plant). The numbers in parentheses are the numbers of isolates recovered.

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b
Titer at 42 days postinoculation expressed as log10 CFU/g (fresh weight). The values in parentheses are the lowest and highest average titers.
c
ND, not determined.

for corn and sorghum endophytes were not significantly tently observed. The colonization levels were maintained
different over the course of the study. throughout all major growth stages of the plants, and the levels
Since bacterial endophytes could be recovered from artifi- of recovery were similar or only slightly reduced over the
cially inoculated corn plants in the greenhouse over an ex- sampling period for 9 of the 13 bacterial strains in each field
tended period of time, another study was conducted in field trial. The average population levels for the remaining four
trials, which provided a fluctuating environment. In two sepa- isolates were less than 2.0 log10 CFU/g (fresh weight). There
rate field trials, five corn endophytes and eight sorghum iso- was no significant difference between corn and sorghum endo-
lates were tested. All of these strains were recovered from phytes in both field trials.
growing dent corn plants through day 71 postinoculation (field Long-range quantitative population data for selected corn
trial I) or day 78 postinoculation (field trial II) (Table 4). (strain CE648) and sorghum (strains SE017 and SE034) endo-
Similar to the greenhouse experiments, the highest coloniza- phytes in greenhouse- and field-grown corn are shown in Fig.
tion levels at both sites were observed on day 1, and at the 1A to C. Isolates CE648 and SE034 displayed the greatest
remaining times moderate levels of colonization were consis- colonization titers for early samples in the greenhouse studies

TABLE 4. Recovery of wild-type agronomic crop endophytic bacteria from dent corn
Corn endophytes Sorghum endophytes
Location Daya No. of Colonization level c
No. of Colonization levelc
isolatesb (avg ⫾ SD) isolatesb (avg ⫾ SD)

Greenhouse
1 9 (9) 6.1 ⫾ 0.1 (4.7–6.8)d,e 8 (8) 5.7 ⫾ 0.1 (4.9–6.7)e
11 9 (9) 5.1 ⫾ 0.2 (4.5–6.3)f 8 (7) 5.7 ⫾ 0.2 (4.9–6.7)f
67 9 (3) 4.4 ⫾ 0.3 (2.6–5.9) 8 (3) 4.3 ⫾ 0.3 (3.5–5.2)

Field trial I
1 5 (5) 5.4 ⫾ 0.2 (4.2–6.6)g 8 (7) 5.0 ⫾ 0.2 (3.8–6.0)g
9 5 (5) 4.1 ⫾ 0.2 (3.4–5.1) 8 (8) 4.2 ⫾ 0.2 (3.4–5.4)h
21 5 (2) 4.0 ⫾ 0.3 (3.5–4.4) 8 (6) 3.8 ⫾ 0.2 (3.0–4.4)
46 5 (4) 3.8 ⫾ 0.2 (2.9–4.7) 8 (8) 3.8 ⫾ 0.2 (2.7–4.7)
71 5 (3) 3.8 ⫾ 0.2 (3.1–4.9) 8 (6) 3.7 ⫾ 0.2 (2.3–4.3)

Field trial II
1 5 (5) 4.5 ⫾ 0.2 (3.4–5.7)i 8 (7) 4.4 ⫾ 0.2 (2.7–5.8)i
14 5 (2) 4.2 ⫾ 0.3 (2.9–5.1) 8 (7) 4.1 ⫾ 0.2 (3.2–4.9)j
26 5 (2) 3.4 ⫾ 0.3 (3.0–3.8) 8 (5) 3.6 ⫾ 0.2 (2.4–4.2)
52 5 (3) 3.8 ⫾ 0.3 (3.0–5.4) 8 (5) 3.8 ⫾ 0.2 (3.4–4.6)
78 5 (2) 3.5 ⫾ 0.3 (2.5–4.2) 8 (5) 3.4 ⫾ 0.2 (2.6–4.3)
a
Number of days between inoculation and plant harvest.
b
Number of isolates tested (inoculated at a level of 7.0 log10 CFU/plant). The numbers in parentheses are the numbers of isolates recovered.
c
Titer for each day expressed as log10 CFU/g (fresh weight). The values in parentheses are the lowest and highest average titers.
d
Significant difference (P ⬍ 0.05) between days 1 and 11.
e
Significant difference (P ⬍ 0.05) between days 1 and 67.
f
Significant difference (P ⬍ 0.05) between days 11 and 67.
g
Significant difference (P ⬍ 0.05) when day 1 was compared to days 9, 21, 46, and 71.
h
Significant difference (P ⬍ 0.05) when day 9 was compared to days 21 and 71.
i
Significant difference (P ⬍ 0.05) when day 1 was compared to days 26, 52, and 78.
j
Significant difference (P ⬍ 0.05) between days 14 and 78.
2204 ZINNIEL ET AL. APPL. ENVIRON. MICROBIOL.

were gram positive, resistant to 50 ␮g of hygromycin per ml,

and susceptible to 10 ␮g of chloramphenicol per ml. Isolate
SG041 was positive for motility and chitin utilization. In addi-
tion, the fatty acid analysis identified the prairie plant endo-
phyte LB030 as Microbacterium esteraromaticum (equivalent to
DSM 8609, IFO 3751, and ATCC 8091), PD039 as Clavibacter
michiganensis subsp. nebraskensis (equivalent to DSM 7483
and ATCC 27794), and SG041 as Curtobacterium flaccumfa-
ciens subsp. flaccumfaciens (equivalent to DSM 20129, IFO
12156, and ATCC 6887). Carbon source utilization tests iden-
tified LB030 as M. testaceum, PD039 as Clavibacter michi-
ganensis subsp. insidiosus (equivalent to DSM 20157 and
ATCC 10253), and SG041 as Curtobacterium citreum (equiva-

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lent to DSM 20528, IFO 12677, and ATCC 15828).
To confirm the results obtained by these procedures, we
analyzed the entire 16S rRNA gene sequence by PCR ampli-
fication using the primer sets described in Materials and Meth-
ods. As determined by this method, the LB030 16S rRNA gene
was 96% homologous to the Microbacterium arabinogalactano-
lyticum type strain DSM 8611 (⫽ IFO 14344 ⫽ ATCC 51926)
16S rRNA gene (39), and the strain LB030 16S rRNA gene
was 96% homologous to the partial 16S rRNA genes of Mi-
crobacterium sp. strain VKM Ac-1807 and Microbacterium
phyllosphaerae. The PD039 16S rRNA gene exhibited 99%
homology to the 16S rRNA gene of Clavibacter michiganensis
type strain DSM 46364 (⫽ IFO 12471) (32), whereas the
SG041 16S rRNA gene exhibited 99% homology to the 16S
rRNA gene of Curtobacterium luteum type strain DSM 20542
(⫽IFO 12676 ⫽ ATCC 15830) (32).
Preliminary taxonomic identification of an additional 52
corn and sorghum endophytes was carried out by performing a
FIG. 1. Persistence of selected endophytic isolates in dent corn. fatty acid analysis. Only 36 of these strains could be classified
Strains CE648 (A), SE017 (B), and SE034 (C) were tested in long- in a genus yielding a similarity index greater than 0.3 with the
range greenhouse studies and two separate field trials using a random- Sherlock Microbial Identification System. The 15 genera iden-
ized complete block design. Strain IFO 12675 (D) is the M. testaceum
type strain, and it was tested only in the greenhouse. Symbols: 䊐, tified were Agrobacterium, Bacillus, Bradyrhizobium, Cellu-
greenhouse; E, field trial I; F, field trial II. The error bars indicate lomonas, Clavibacter, Corynebacterium, Enterobacter, Erwinia,
standard deviations (n ⱖ 3). The minimum detection limit was 102 Escherichia, Klebsiella, Microbacterium, Micrococcus, Pseudo-
CFU/g (fresh weight). monas, Rothia, and Xanthomonas. Of these, Bacillus, Coryne-
bacterium, and Microbacterium were the most prevalent gen-
era, with eight, five, and nine assigned isolates, respectively.
(Fig. 1A and C). The levels for all of the isolates in one or both The three corn and sorghum endophytes (CE648, SE017,
field trials were 3.1 log10 CFU/g (fresh weight) or greater after and SE034) were gram positive, resistant to 50 ␮g of hygro-
at least 50 days (Fig. 1A to C). mycin per ml and 50 ␮g of gentamicin per ml, and susceptible
Considering the potential use of endophytes exhibiting se- to 10 ␮g of chloramphenicol per ml. Isolate SE034 was positive
lectable phenotypes in other applications (6, 37), we isolated for motility and chitin utilization, while CE648 was positive
spontaneous kanamycin-, rifampin-, and tetracycline-resistant only for chitin utilization. Fatty acid analysis identified the
mutants from the corn and sorghum endophytes that exhibited agronomic crop endophyte CE648 as Microbacterium arbore-
the highest colonization levels. These mutants were tested in scens (equivalent to DSM 20754, IFO 3750, and ATCC 4358),
long-range colonization studies performed with dent corn in SE017 as Cellulomonas flavigena (equivalent to DSM 20109T,
both greenhouse studies and field trials. The results, including IFO 3754, and ATCC 482), and SE034 as M. testaceum (equiv-
the levels of colonization, were similar to those obtained with alent to DSM 20166, IFO 12675, and ATCC 15829). The car-
the corresponding wild-type strains, but there were some dif- bon source utilization analysis identified all three corn and
ferences (data not shown). Levels for antibiotic-resistant iso- sorghum endophytes as M. testaceum. Similarly, the CE648,
lates were significantly lower than those for wild-type isolates SE017, and SE034 16S rRNA genes exhibited 99% homology
during field trial I (days 9, 21, and 46) and during field trial II to the 16S rRNA gene of the M. testaceum type strain (32). A
(day 14). phenogram reflecting the relationships among the endophytic
Taxonomic identification of selected bacterial endophytes. and candidate strains used in all of the analyses described
A number of agronomic crop and prairie plant endophytes above was constructed based on the 16S rRNA gene sequences
were selected for taxonomic studies based on the colonization as described in Materials and Methods (Fig. 2). Because the
studies described above. Isolates LB030, PD039, and SG041 corn and sorghum endophytes were all identified as M. testa-
VOL. 68, 2002 ENDOPHYTIC BACTERIA IN PLANTS 2205

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FIG. 2. Phenogram expressing the relationships of identified bacterial endophytes to taxonomically similar microorganisms based on the 16S
rRNA gene sequences. The unrooted phenogram was constructed as described in Materials and Methods. The GenBank accession number is given
in parentheses for each organism. The positions of the endophyte strains are based on the best match for genus and species. The numbers at the
nodes are bootstrap values based on 100 replications. The 16S rRNA gene sequence of Bacillus subtilis was utilized as an outgroup.

ceum isolates by the 16S rRNA gene sequence analysis, we moderate to high levels through 42 days postinoculation (Table
tested the type strain of M. testaceum in long-range greenhouse 2). To our knowledge, this is the first experimental greenhouse
colonization studies. The results demonstrated that this organ- study of colonization of prairie plants by bacterial endophytes.
ism was able to colonize dent corn at levels similar to the levels However, other workers have conducted similar studies in
of colonization by the agronomic crop endophyte strains iso- growth chambers. For example, the rhizobacterium Pseudomo-
lated in our study (Fig. 1D). nas aureofaciens was inoculated and was recovered after 29
days from tall fescue leaves and pea and bean stems at a level
of 2.3 log10 CFU/g (fresh weight) (24).
DISCUSSION
The ability of corn and sorghum endophytes to colonize and
Our research goals were to survey agronomic crops and persist in dent corn and sorghum was initially shown by using
prairie plants for the presence of endophytic bacteria and to seedlings (Table 2). Based on an inoculum level of 6.0 log10
determine their phenotypic properties, their taxonomic posi- CFU/plant, some multiplication of the endophytes occurred in
tions, and their colonization levels in experimentally inocu- many plants. This is the first demonstration of endophytic
lated plants. In this study, we isolated several hundred bacte- colonization of experimentally inoculated sorghum seedlings.
rial strains from dent corn, sorghum, soybean, wheat, perennial A subset of endophytes identified in the growth room studies
grasses, forbs, legumes, and prairie wildflowers (Table 1). Sim- that yielded high levels of colonization were analyzed in more
ilarly, other workers have reported isolation of indigenous comprehensive long-range greenhouse studies and field trials
endophytic bacteria from yellow dent type corn (7), sweet corn in which dent corn was used as the host. As expected, the
(12, 28), and alfalfa (14). To our knowledge, our study is the colonization rates observed in the greenhouse were higher
first to describe indigenous bacterial endophytes isolated from than those achieved in the field. In the field, there is increased
sorghum, soybean, wheat, and native perennial plants. There microbial competition, the cultural practices, such as irrigation,
appears to be significant variation in the types of indigenous are less consistent, and there may be unfavorable environmen-
bacteria isolated from diverse host plant species. Several fac- tal conditions, such as high temperatures. In contrast, the ster-
tors may explain these differences, including host specificity, ile seedling inoculation experiments resulted in population lev-
geographical distribution, plant age, and tissue type (21). els that were usually 10-fold higher than the levels in the
In our colonization studies of prairie plant endophytes in the greenhouse. Most of the corn and sorghum endophytes per-
greenhouse, we demonstrated that most of the introduced en- sisted at significant average colonization levels ranging be-
dophytic strains could recolonize the original plant host at tween 3.4 and 6.1 log10 CFU/g (fresh weight) in the greenhouse
2206 ZINNIEL ET AL. APPL. ENVIRON. MICROBIOL.

and fields for up to 67 and 78 days, respectively (Table 4 and except SE017, which was identified as a Cellulomonas strain by
Fig. 1). Antibiotic-resistant mutants obtained from selected fatty acid analysis and as a Microbacterium strain by the other
strains of these endophytes could also colonize plants at levels two methods. The endophytes PD039 (Clavibacter michiganen-
similar to the levels of wild-type strains in these greenhouse sis) and SE034 (M. testaceum) were each identified as the same
and field experiments. In this context, similar results were organism at the genus and species levels by all tests.
obtained in other plant species for antibiotic-resistant mutants, The close association observed between soil and plant envi-
as reviewed by Kobayashi and Palumbo (21), for Bacillus sub- ronments suggests a potential endophytic role for Cellulomo-
tilis, Erwinia sp., and Pseudomonas. In one of these studies, nas and Microbacterium strains. Our studies identified for the
Frommel et al. (13) obtained higher average population levels first time the endophytic nature of M. testaceum for various
of rifampin- and nalidixic acid-resistant mutants of Pseudomo- plant hosts. Cellulomonas flavigena has been isolated as an
nas sp. in potato roots in both greenhouse studies and field epiphytic bacterium from the surfaces of olive leaves (10),
trials. This result may be a reflection of the ability of Pseudo- while M. testaceum was isolated from Chinese rice paddies
monas sp. to establish high population densities in the rhizo- (23). Researchers have identified microbial antagonists be-

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sphere. longing to the genus Microbacterium that decrease nematode
Other investigators have performed bacterial endophyte col- populations for up to 96 days in the greenhouse and through-
onization studies with corn or other agronomic crops, obtain- out the following growing season in field trials (18, 46). The
ing levels comparable to those assessed here. Lamb et al. (24) chitinolytic microorganism M. testaceum has been isolated
tested the ability of the rhizobacterium P. aureofaciens to col- from cotton rhizospheres only when chitin has been added to
onize corn, wheat, oats, and cotton. Microorganisms were re- the soil (18). A second coryneform bacterium, M. esteraromati-
covered at levels of 2.5 to 4.7 log10 CFU/g (fresh weight) from cum, has been isolated from soybean rhizospheres after use of
the aerial portions of these plants. Similarly, inoculation of a nematicide-producing velvetbean cropping system (46). Cur-
Clavibacter xyli subsp. cynodontis onto corn seeds by using a tobacterium citreum and Curtobacterium luteum were originally
pressure bomb resulted in recovery levels as high as 9.0 log10 isolated from Chinese rice paddies (23). Strains of Curtobac-
CFU/g (fresh weight) of tissue (11). In contrast, there have terium flaccumfaciens cause bacterial diseases (47) but also
been few population studies of the levels of recovery and col- have been recovered from the phyllosphere of wheat (26) and
onization of indigenous endophytes in the aerial portions of have been shown to be a biocontrol agent for cucumber (33).
corn. McInroy and Kloepper (27) isolated naturally occurring These three bacterial species have been isolated as endophytes
endophytic bacteria from sweet corn grown in the greenhouse from red clover nodules, and Curtobacterium luteum consis-
and field and reported population levels ranging from 3.0 to 7.0 tently promotes plant growth alone or in combination with
log10 CFU/g (fresh weight). Fisher et al. (12) recovered bac- Rhizobium spp. (40). The Clavibacter michiganensis group con-
teria from field-grown sweet corn and reported colonization tains subspecies that can cause diseases in their natural hosts,
levels of 2.3 to 6.5 log10 CFU/g (wet weight) of tissue. From the such as bacterial wilt of alfalfa, bacterial canker of tomato and
latter reports and our studies, it is clear that indigenous bac- pepper, and Goss’s wilt of corn (47). Naturally occurring non-
terial endophytes can be isolated from corn plants throughout pathogenic strains, such as our strain PD039, have not been
the entire growing season in many different environments with reported previously.
different corn varieties. In addition, the population levels for Based on the 16S rRNA gene sequence analysis results, we
endophytes (single or mixed) are fairly consistent and are be- constructed a phenogram for the six endophytes mentioned
tween 2.0 and 7.0 log10 CFU/g of tissue. above and related microorganisms (Fig. 2). Endophytes SE017,
In our host range study, endophytes obtained from the SE034, CE648, and LB030 fall into the Microbacterium group
monocots corn and sorghum were shown to colonize and per- containing M. testaceum, M. arabinogalactanolyticum, M. arbo-
sist in both monocot and dicot plant species (Table 3). The rescens, and M. esteraromaticum (39, 42, 43, 52). The first three
highest endophyte population levels were obtained for the strains were definitively identified as M. testaceum, whereas the
monocot hosts (onion and wheat). Other workers have also fourth isolate could not be clearly identified to the species level.
looked at the recovery of endophytes from monocot plants Endophyte PD039 belongs to the genus Clavibacter and is most
(corn and banana) after the organisms were originally isolated closely related to the species Clavibacter michiganensis, including
from monocot species (21). The previous studies include En- Clavibacter michiganensis subsp. nebraskensis and Clavibacter
terobacter cloacae, Burkholderia cepacia, and Clavibacter xyli michiganensis subsp. insidiosus (32). Endophyte SG041 fits well
subsp. cynodontis isolated from many different plant species, into the Curtobacterium group consisting of Curtobacterium cit-
suggesting that these bacteria have developed an evolutionary reum, Curtobacterium flaccumfaciens, and Curtobacterium luteum
niche within plants. (9, 32).
Six endophytes with the most promising levels of coloniza- Modification of plants to obtain organisms with improved
tion and the ability to persist in a range of host plants were genetic capabilities and tolerance to different environmental
chosen for taxonomic identification based on 16S rRNA gene conditions is generally carried out by plant breeding and by
sequence, commercial fatty acid, and carbon utilization analy- integrating foreign DNA into plant genomes to produce trans-
ses. Tang et al. (44) compared these methods for identification genic plants (53). Although successful for certain plants, these
of gram-negative bacilli to the genus and species levels and methods are costly, are dependent on the plant variety being
reported that 16S rRNA gene sequence analysis was 10 and studied, and take several years to reach market. As an alter-
20% more accurate than the commercial fatty acid and carbon native approach, beneficial endophytes have been used to ex-
utilization analysis systems, respectively. In our study, all anal- press and secrete useful products without requiring integration
yses resulted in the same genus level identity for all endophytes of foreign DNA into the plant genome. Endophytic bacteria
VOL. 68, 2002 ENDOPHYTIC BACTERIA IN PLANTS 2207

have a multitude of applications that enhance agricultural pro- 16. Gross, D. C., and A. K. Vidaver. 1979. A selective medium for isolation of
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phytohormones (2), increase rice production by increasing 17. Hallmann, J., A. Quadt-Hallmann, W. F. Mahaffee, and J. W. Kloepper.
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914.
contribute to corn pest management (11), fix nitrogen in rice 18. Hallmann, J., R. Rodriguez-Kabana, and J. W. Kloepper. 1999. Chitin-
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and increase potato tuber formation under heat stress condi- within roots of cotton in relation to nematode control. Soil Biol. Biochem.
31:551–560.
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for assistance with statistical analysis of data concerning the isolation soybeans: population levels of Pseudomonas glycinea in relation to symptom
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