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Environmental and Experimental Botany

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Endophytic bacteria in cacti seeds can improve the development of cactus

seedlings
M. Esther Puente a , Ching Y. Li b,1 , Yoav Bashan a,c,
a

Environmental Microbiology Group, Northwestern Center for Biological Research (CIBNOR), Mar Bermejo 195, Colonia Playa Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico
Pacic Northwest Research Station, USDA Forest Service, 3200 SW Jefferson Way, Corvallis, OR 97331, USA
c
Department of Soil, Water and Environmental Science, 429 Shantz Building #38, The University of Arizona, Tucson, AZ 85721, USA
b

a r t i c l e

i n f o

Article history:
Received 20 August 2008
Received in revised form 3 February 2009
Accepted 21 April 2009
Keywords:
Bacillus
Cactus
Cardon
Desert
Rock degradation
Nitrogen xation
Pachycereus
Phosphate solubilization
Rock weathering
Soil formation

a b s t r a c t
A plantbacterium association between the giant cardon cactus Pachycereus pringlei and endophytic bacteria help seedlings establish and grow on barren rock. This cactus, together with other desert plants, is
responsible for weathering ancient lava ows in the Baja California Peninsula of Mexico. When cardon
seeds are inoculated with endophytic bacteria, the seedlings grow in pulverized rock for at least a year
without fertilization and without showing distress. The bacteriaplant association released signicant
amounts of necessary nutrients from the substrate. When endophytic bacteria were eliminated from the
seeds by antibiotics, development of seedlings stopped. In complementary experiments of sterile seeds
inoculated with the same endophytic bacteria, plant growth was restored. This study and the previous
one show that, under extreme environmental conditions, a symbiotic relationship is present between
endophytic bacteria and their cactus host.
2009 Elsevier B.V. All rights reserved.

1. Introduction
Several species of desert plants, mainly cacti, grow without soil
on rocky cliffs, large rocks, and ancient lava ows in hot desert
areas of the Baja California Peninsula of Mexico where weathering
is not apparent (Bashan et al., 2002, 2006). Higher plants are known
to considerably affect the kinetics of dissolution of basalt in other
environments (Hinsinger et al., 2001). Chemical weathering of lava
ows beneath mature forests continues unabated for thousands of
years after initial colonization (Cochran and Berner, 1996). In Iceland, the rate of weathering of rock by higher plants is two to ve
times higher in vegetated areas than in barren areas (Moulton and
Berner, 1998). Involvement of microorganisms in these processes
is inherent because they are widely present in bulk soil (Berthelin
et al., 1991). Mycorrhizal fungi in European coniferous forests that
grow on shallow granite rock are able to penetrate and dissolve the
rocks. Dissolved products are translocated by the host plant roots,

Corresponding author at: Environmental Microbiology Group, Northwestern

Center for Biological Research (CIBNOR), Mar Bermejo 195, Colonia Playa Palo de
Santa Rita, La Paz, B.C.S. 23096, Mexico. Tel.: +52 612 123 8484x3668;
fax: +52 612 125 4710.
E-mail address: bashan@cals.arizona.edu (Y. Bashan).
1
Retired.
0098-8472/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.envexpbot.2009.04.007

bypassing the soil solution and bypassing competition for nutrient

uptake by other organisms (van Breemen et al., 2000). Frankia and
Alpova diplophloeus assisted growth, nitrogen xation, and mineral
acquisition by Alnus tenuifolia (Yamanaka et al., 2003).
In deserts, the rhizoplane population of rock-dwelling cacti contains many plant growth promotion traits, such as a capacity to
dissolve minerals, x nitrogen, and promote plant growth (Puente
et al., 2004a,b). These plants contain endophytic bacteria with similar growth-promoting traits (Puente et al., 2009).
This study explored the potential of these endophytic bacteria
to promote plant growth of cardon cacti. We hypothesized that
the endophytes are essential for normal growth of cactus and, if
removed, plant growth is impaired.
2. Materials and methods
2.1. Organisms
Seeds of the cardon cactus (Pachycereus pringlei [S. Watson] Britton & Rose) were used in all experiments. The following strains of
endophytic bacteria were used and their nucleotide sequences were
deposited in GenBank: EF123226 Klebsiella sp. SENDO 1, EF123227
Acinetobacter sp. SENDO 1, EF123229 Pseudomonas sp. SENDO 2,
EF123230 Bacillus sp. SENDO 6, EF123231 Klebsiella sp. SENDO

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2, EF123233 Staphylococcus sp. SENDO 2 (Puente et al., 2009).

Two positive controls, the plant growth-promoting bacteria (PGPB)
Pseudomonas putida R-20 (Meyer and Linderman, 1986) and the
PGPB Azospirillum brasilense Cd, ATCC 29710 were also used. Additionally, several mixtures of bacteria were tested: each of the six
endophytes listed above, endophytes with each of the PGPB, and
endophytes with both PGPBs.
2.2. Inoculation of cardon cacti grown in pulverized rock with
endophytic isolates
Ancient extrusive igneous rocks (lava ows) were subjected
to bacterial weathering experiments after being oven-sterilized
(250 C, overnight), pulverized in a mill, and sieved to obtain <90m particles (rock our). Rock our (4 g) was mixed with 23 g
pulverized perlite and placed in small black pots. For a negative control, pots were lled with 27 g perlite. Cardon seeds were
washed thoroughly with 2% detergent (Tween 20) for 10 min to
remove residual dust. Seed surfaces were disinfected with 3%
commercial hypochlorite bleach for 5 min and then rinsed continuously for 10 min with sterile distilled water. Seeds were inoculated
with a selected species of bacteria by a standard vacuum inltration technique (Puente and Bashan, 1993) at a concentration of
1 106 CFU ml1 . Briey, seeds were inoculated by dipping them
in bacterial suspensions for 5 min under a vacuum of 600 mm Hg.
Then, the vacuum was released abruptly, allowing the bacteria to
penetrate seed cavities that were previously lled with air.
Ten seeds were placed on the surface of the substrate in each
pot, which had been irrigated with 50 ml distilled water, and
then covered with a 5-mm layer of dry substrate. The pots were
incubated in a growth chamber (Biotronette Mark III, Barnstead
International, Dubuque, IA) at 30 2 C under light intensity of
70 mol photons m2 s1 for 12 h for 12 months. Pots were irrigated every 15 days with 25 ml basal Hoaglands nutrient solution
without phosphorus and nitrogen (for the two strains of Klebsiella
sp., mixture of endophytic bacteria, and controls) or with nitrogen,
but without phosphorus (for Staphylococcus sp., Acinetobacter sp.,
two strains for Pseudomonas sp.). The perlite negative controls were
also irrigated with phosphorus. The positive control plants were
inoculated with a phosphate solubilizing bacterium, P. putida, and
grown in perlite only, irrigated with Hoaglands nutrient solution
containing nitrogen, while those inoculated with the diazotroph
A. brasilense and grown in perlite were irrigated with Hoaglands
nutrient solution with phosphorus but without nitrogen. Uninoculated plants served as controls. One additional positive control was
irrigated with Hoaglands nutrient solution without phosphorus or
nitrogen and all bacteria and the other positive control was irrigated with complete Hoaglands nutrient solution. Before and after
plants were grown in the rock our with perlite, the rock was analyzed for P2 O5 , total phosphorus, K2 O, MgO, and Fe2 O3 , as described
in the previous paper (Puente et al., 2009). At the end of the experiment, plants were extracted and photographed. Height and volume
(Bashan et al., 1999) and root length and dry weight were measured.
The drying oven was set at 50 C for 120 h. Total nitrogen content of
the plants was measured by an automatic, micro-Kjeldahl method
after digestion of the samples (Digestion System 12.1009 and Kjeltec
Auto 1030 Analyzer, Tecator, Hgans, Sweden).
2.3. Elimination of endophytic bacteria in seeds treated with
antibiotics
Since all batches of cardon seeds collected from wild plants contained bacterial endophytes, an attempt was made to eliminate
them and produce endophyte-free seedlings by soaking seeds in the
following antibiotic solutions (g ml1 ) for 20 min: chloramphenicol (500); streptomycin sulfate (200), tetracycline (12), penicillin G

403

(500), rifampicin (150), and mixtures of all antibiotics at the above

concentrations. To assay for sterile seeds, 1 g of surface-disinfected
seeds was pulverized with a pestle and mortar in 10 ml PBS. The
homogenized slurry was transferred to tryptic soy broth (50 ml)
and incubated with agitation (120 rpm) for 24 h at 30 C. Then, 1 ml
aliquots were taken from each suspension of bacteria and assayed
for cultivable bacteria by the plate count method, total bacteria by
the FITC method, and viable bacteria by the FDA method (Puente
et al., 2009). For comparison, 1 g of seeds was treated with 1 ml of
each of the antibiotics in test tubes and incubated for 3 h under the
same conditions. The same assays for bacteria were made with the
three methods listed here to decide which antibiotic (or combination) worked best. Seeds of lodgepole pine Pinus contorta Dougl. ex
Loud., which are susceptible to antibiotic applications, served as a
control for the germination tests.
2.4. Field emission scanning electron microscopy
For this test, root samples (0.51.5 cm long) were xed with
5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) immediately after eld sampling and sliced in half with a sterile razor.
The following day, roots were rinsed in cacodylate buffer, dehydrated in a series of increasing ethanol concentrations from 30 to
100% for 20 min each, and dehydrated with isoamyl acetate. After
dehydration, samples were dried with CO2 in a critical point dryer
(Samdri-PVT-3B, Tousimis Research, Rockville, MD). The dried samples were xed to stubs with double-sided adhesive tape and coated
with 30 nm 60:40%, gold:palladium alloy foil in a sputter coater
(Edwards S150B) and then examined at 7 kV with a eld emission
scanning electron microscope (AmRay 3300FE, Advanced Metals
Research, Bedford, MA, USA).
2.5. Mineral analyses
After cultivation trials lasting 1 year, plants were removed and
the remaining substrate was analyzed for minerals (K2 O, Fe2 O3 ,
and MgO) by EPA Method 3015-microwave digestion (nitric acid)
(Kingston, 1994) with an atomic absorption spectrometer (GBC Scientic Equipment, Dandenong, Victoria, Australia). Concentrations
of phosphate (P2 O5 ) were determined according to Jackson (1958).
Substrate without plants but under the same conditions served as
control.
2.6. Experimental design and statistical analysis
Plants were inoculated in 10 pot replicates thinned to 3 seedlings
per pot. Pots were distributed randomly in the growth chamber, and
occasionally rearranged during 12 months of cultivation. Germination tests were made with 5 replicates, where 1 Petri dish containing
25 seeds served as a replicate. Triplicate samples were analytically
assayed. One-way ANOVA, followed by Tukeys ad-hoc analysis or
Students t-test at P < 0.05 was made with statistical software (Statistica vers. 6, StatSoft, Tulsa, OK). Numerical data are accompanied
by standard errors.
3. Results
3.1. Production of endophyte-free seedlings and the effect of
endophytes on seedling development
To evaluate the importance of endophytic bacteria in seeds
developing into seedlings, seeds were treated alone and with combinations of antibiotics to eliminate the endophytic populations.
While each antibiotic treatment reduced the initial endophytic
population (all values in bacteria g1 dw seeds), from 760 106 to
>1 106 , only the combination of cholramphenicol and tetracycline

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M.E. Puente et al. / Environmental and Experimental Botany 66 (2009) 402408

Fig. 1. (A) Viable endophytic bacteria count (by FDA) after treatment with different antibiotics. (B) The same counts under different concentrations of an antibiotic mixture
(mixture contained: chloramphenicol 500 g ml1 and tetracycline 12 g ml1 ). (C) Effect of different concentrations of an antibiotic mixture on germination of cardon seeds.
Effect of two concentrations of an antibiotic mixture on dry weight (D), root length (E), and height (F) of cardon seedlings growing for 10 days in pulverized igneous rock
substrate after treatment. Columns denoted by a different letter, in each subgure, differ signicantly at P < 0.05 by one-way ANOVA. Bars represent SE. The absence of SE
indicates a negligible value. The experiment was repeated ve times.

initially reduced bacteria to 1 103 (Fig. 1A). Therefore, a mixture

of the two antibiotics was further evaluated for later seed treatments. Dilutions of the antibiotic mixture below 50% of the original
concentration failed to eliminate endophytes. Concentrations >50%
completely eliminated endophytes (Fig. 1B). When germination
was evaluated after 11 days, antibiotic mixtures at any dilution
(2.5100%) had no deleterious effect on cactus seed germination
(Fig. 1C) and did not affect germination of the pine seeds (P. contorta)
used as the antibiotic-sensitive control. Without antibiotics treatment (control), 77 1% of the seeds germinated; at 100% antibiotic
application, 74 3.46% germinated. Germination rates were similar
at six other dilutions, but none of the differences were statistically
signicant. However, very young seedlings grown without endophytes were signicantly smaller in dry weight, root length, and

Fig. 2. Growth promotion of cardon plants growing in volcanic rock for 12 months
and inoculated with the endophytic bacteria Pseudomonas sp. SENDO 2 (A) and Bacillus sp. SENDO 6 (C) compared to plants growing in the same substrate without
inoculation with endophytic bacteria. The experiment was repeated twice.

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Fig. 3. Effects of six endophytic bacteria, two control PGPBs, and one mixture of endophytic bacteria on promotion of growth of giant cardon cactus seedlings (dry weight,
volume, height, root length, and nitrogen content) growing in pulverized igneous rock supplemented with perlite (AE) or exclusively on perlite (FJ) for 12 months after
inoculation of seeds. Columns denoted by a different letter in each subgure differ signicantly at P < 0.05 by one-way ANOVA. Bars represent SE. The experiment was repeated
twice.

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height (Fig. 1DF). Germinating seeds containing endophytes and

the seedlings were irrigated with the successful antibiotic treatment over the following 8 months and showed no negative effects
on plant growth (data not shown).
3.2. Effects of inoculation with endophytic bacteria on the growth
of cardon seedlings
Disinfected cardon seeds were tested with six species of endophytic bacteria and a mixture of all strains. Two known PGPB
strains were used as positive controls. These inoculation treatments signicantly changed several growth parameters during the
experiment lasting 1 year (Fig. 2). Dry weight, volume, root length,
and height of inoculated plants were signicantly greater in most
cases (Fig. 3AJ). The four growth parameters for plants growing in pulverized rock substrate and inoculated with endophytic
bacteria were signicantly greater than plants serving as controls.
The results of inoculation with the positive control PGPB P. putida
have the same trends, but smaller, to those of the endophyic bacteria (Fig. 3AD), but inoculation with the other positive control
PGPB A. brasilense promoted only dry weight and root length over
controls that were not inoculated (Fig. 3A and C). We could not
determine if the strains persisted on the roots after the 1-year trial
because molecular markers were not available to identify them
at the time of the experiments. Three endophytic bacteria, Bacillus sp. SENDO 6, Acinetobacter sp. SENDO 1, and Pseudomonas sp.
SENDO 2 performed better in the rock-perlite substrate than the
other three bacteria, whereas the mixture of all endophytes, while
performing better than the controls that were not inoculated, was
inferior to single-endophytic inoculations for most tested parameters.
One year after inoculation, plants inoculated with any of
the six endophytes or the control PGPB endophytes signicantly
increased total nitrogen content of the plants grown in pulverized rock-perlite, while the mixture of the six endophytes and
the PGPB did not (Fig. 3I). When plants were grown only in
perlite, all bacteria increased the nitrogen content of the plants
(Fig. 3J).
Histological (not shown) and SEM studies of young cardon cacti
grown from endophyte-free seeds, but inoculated with the Klebsiella sp. SENDO 2 and Bacillus sp. SENDO 6 resulted in root and
stem colonization by endophytes during the year after inoculation
and rst detected after 30 days (Fig. 4). Seedlings that had not
been inoculated remained axenic for the duration of the 30-day
experiment.
3.3. Weathering of rock minerals after growth of cardon seedlings
in substrate
After growing for 1 year in pulverized rock-perlite substrate,
inoculated cardon cacti had mineralized signicant quantities of
several minerals essential to plant growth in the substrate. Plants
inoculated with any of the bacteria species removed more P2 O5
from the substrate than uninoculated plants; those inoculated with
Klebsiella sp. SENDO 2 (30%), Bacillus sp. SENDO 6 (29%), Pseudomonas sp. SENDO 2 removed the most (26%) phosphate (Fig. 5).
Plants inoculated with any of the endophytic bacterial species
removed Fe2 O3 at higher levels than uninoculated plants, where
Klebsiella sp. SENDO 2, Staphylococcus sp. SENDO 2, and Pseudomonas sp. SENDO 2 removed the most (34%), while the two
control PGPB did not increase the plants ability to remove more
Fe2 O3 than uninoculated plants (Fig. 5B). Plants inoculated with
several of the bacteria removed signicant amounts of K2 O; Pseudomonas sp. SENDO 2, and the mixture of all endophytes removed
the largest amount of potassium (32%) (Fig. 5C). All plants inoculated with endophytes removed signicantly more MgO than

Fig. 4. Colonization by endophytic bacteria of the interior connecting tissue between

the roots and the stem of cardon plantlets growing from seeds free of endophytes and
articially inoculated with the endophytes Klebsiella sp. SENDO 2 (A) and Bacillus sp.
SENDO 6 (B). (C) Control, uninoculated plants. Arrows indicate bacteria and possible
aggregates of bacteria.

uninoculated plants; those with Klebsiella sp. SENDO 1 and SENDO

2 and Bacillus sp. SENDO 6 removed the most (63%) (Fig. 5D). Plants
inoculated with the two control PGPB were less efcient at removing P2 O5 , Fe2 O3 , and MgO than plants inoculated with endophytic
bacteria, but were equally efcient as the endophytic bacteria at
removing K2 O (Fig. 5C). The pH of the substrate was lowered by the
presence of cacti, but was signicantly reduced if the plants were
inoculated with the mixture of endophytes (Fig. 5E).
4. Discussion
Plant growth-promoting bacteria (PGPB; Bashan and Holguin,
1998) are commonplace in agriculture (Bashan and de-Bashan,
2005), but are rarely reported for wild plants of environmental
importance (Bacilio et al., 2006). Recently, involvement of bacteria
in phyto-remediation, in general (Lodewyckx et al., 2001, 2002a;
Glick, 2003, 2004; Chaudhry et al., 2005; Reed et al., 2005; Bashan
et al., 2008), and more specically, aiding plants growing on mine
tailing was proposed (Grandlic et al., 2008; Mendez et al., 2007;
Rosario et al., 2007). Further, endophytic bacteria are well known
in crop plants (Hallmann et al., 1997; Lodewyckx et al., 2002b), but
largely has not be investigated in wild plants.

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Fig. 5. (AD) Removal of P2 O5 , K2 O, Fe2 O3 , and MgO from pulverized rock substrate in which cardon cacti, inoculated with six endophytic bacteria, two control PGPBs, and
one mixture of endophytic bacteria, were grown. Number at each subgure represents the quantity of each of the four minerals in the pulverized rock. Results are presented as
percentage of depletion of the element. (E) pH changes of substrate of pulverized volcanic rock plus perlite by cactus seedlings inoculated with either a mixture of endophytic
bacteria or a control of a PGPB after 1 year of cultivation. Columns denoted by different letters in each subgure differ signicantly at P < 0.05 by one-way ANOVA. Bars
represent SE. The absence of SE indicates a negligible value. The experiment was repeated twice.

In desert plants, agricultural PGPB of the genus Azospirillum

promote the growth of seedlings of the giant cardon cactus and
several other cactus species, a process that provides considerable
soil stabilization in deserts (Bacilio et al., 2006; Puente and Bashan,
1993; Bashan et al., 1999, 2006; Carrillo-Garcia et al., 2000). Several
rhizoplane bacteria isolated from roots of cacti growing in rocky
substrates also promote establishment of cardons (Puente et al.,
2004b). Our working hypothesis was that endophytic bacteria colonizing the interior of almost every cardon plant have PGPB traits
(Puente et al., 2009) and therefore promotes the growth of these
plants, a process reported for agricultural crops (Lodewyckx et al.,
2002b). Furthermore, certain bacteria are essential for the growth
of these cacti under harsh substrate conditions, such as rocks and
rocky surfaces.
Direct evidence that endophytic bacteria promote plant growth
came from crop studies, including potato and clover (for review
Bashan and de-Bashan, 2005). Studies showed that 1021% of the
bacteria recovered from potato tuber bers, for example, are PGPB,
and upon inoculation, promoted plant growth (Sturz, 1995; Sturz
et al., 1998). When slow-growing cardon seedlings were inoculated
with any of six potential endophytic PGPB and grown for 12 months,
signicantly increased growth occurred relative to the controls.
For example, plant volume, which is a critical parameter for cactus survival during the rst year, increased (Puente and Bashan,
1993; Bashan et al., 1999). All inoculated plants, regardless of the
bacteria used, grew much better and were more capable of extracting essential inorganic minerals from the pulverized rock substrate
than uninoculated controls. The breakdown of essential plant elements from rock minerals by inoculated plants can be attributed to
solubilizing activity of the bacteria that colonize the cactus roots

and the root exudates containing organic acids (Lynch and Whipps,
1990; Carrillo et al., 2002). Additionally, nonsymbiotic microora
are known to increase the rate of absorption of Ca and Mg by plants
(Berthelin and Leyval, 1982; Berthelin et al., 1991). The exact source
of nitrogen used by the plants was not determined. However, several of the endophytic bacteria used were diazotrophs (Puente et
al., 2009).
Minerals from igneous and metamorphic rocks contain most
of the nutrients required by higher plants for normal growth
and development. Geochemical studies of the breakdown of rock
minerals and formation of clay minerals have explained the general reaction pathways by which nutrients are released, which is
enhanced by disequilibrium between the soil solution and mineral surfaces through the removal of ions by processes such as
leaching and nutrient uptake. Rhizosphere processes and other biological activity further enhances breakdown of minerals through
the release of hydrogen ions and complex organic compounds that
react with mineral surfaces (Harley and Gilkes, 2000). The bacteria
used in this study have these abilities (Puente et al., 2009).
Taken together, these two papers raise questions for future
research: (1) what is the evolutionary signicance of such associations? (2) How does the extra genetic diversity of endophytes
contribute to better plant growth in ways that the plant genotype
could not satisfy by itself? (3) Are endophytes inoculated into plants
also colonize the rhizosphere, which is essential for accelerating
biological weathering of rocks?
In summary, this study indicates that endophytic bacteria
are essential for the development of cardon seedlings in rocky
substrates and promote growth of cacti in soilless environments.

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Acknowledgements
We thank Al Soeldner at Oregon State University, Corvallis, Oregon, Maria del Carmen Rodrguez, and Ariel Cruz at CIBNOR for
assisting in histology, image analysis, and electron microscopy;

Baudilio Acosta and Griselda Pena-Armenta

for mineral analysis,
for phosphorus analysis, and Sonia Rocha and
Manuel Trasvina
Dolores Rondero for nitrogen analysis, all at CIBNOR. Ira Fogel at
CIBNOR provided editorial clarication. Yoav Bashan participated
in this study in the memory of the late Avner Bashan of Israel. This
work was supported by Consejo Nacional de Ciencia y Tecnologia
of Mexico (CONACYT, project 50052-Z), Pacic Northwest Research
Station of the USDA Forest Service in Corvallis, Oregon, USA, and
the Bashan Foundation in Oregon, USA.
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