ORIGINAL PAPER
Isolation of micropropagated strawberry endophytic bacteria
and assessment of their potential for plant growth promotion
Armando C. F. Dias Æ Francisco E. C. Costa Æ Fernando D. Andreote Æ
Paulo T. Lacava Æ Manoel A. Teixeira Æ Laura C. Assumpc¸a
˜
o Æ
Welington L. Arau
´
jo Æ Joa
˜
o L. Azevedo Æ Itamar S. Melo
Received: 13 June 2008 / Accepted: 2 October 2008 / Published online: 19 October 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Twenty endophytic bacteria were isolated from
the meristematic tissues of three varieties of strawberry
cultivated in vitro, and further identified, by FAME profile,
into the genera Bacillus and Sphingopyxis. The strains were
also characterized according to indole acetic acid produc-
tion, phosphate solubilization and potential for plant
growth promotion. Results showed that 15 strains produced
high levels of IAA and all 20 showed potential for solu-
bilizing inorganic phosphate. Plant growth promotion
evaluated under greenhouse conditions revealed the ability
of the strains to enhance the root number, length and dry
weight and also the leaf number, petiole length and dry
weight of the aerial portion. Seven Bacillus spp. strains
promoted root development and one strain of Sphingopyxis
sp. promoted the development of plant shoots. The plant
growth promotion showed to be correlated to IAA pro-
duction and phosphate solubilization. The data also
suggested that bacterial effects could potentially be har-
nessed to promote plant growth during seedling
acclimatization in strawberry.
Keywords Auxin production Phosphate solubilization
In vitro cultivation Fragaria ananassa
Introduction
The brazilian states of Sa
˜
o Paulo and Minas Gerais are the
biggest producers of strawberries (Fragaria ananassa)in
Latin America (Botelho 1999). Brazilian strawberry crops
are based in the cultivation of plants obtained from plant
tissue cultures, using somatic embryogenesis (Williams
and Maheswaran 1986; Smy
´
kal et al. 2007). This technique
makes it possible to produce a great number of clones, free
of pathogenic fungi and bacteria (Siragusa et al. 2007).
However, the process of explant disinfection might also
eliminate non-pathogenic microorganisms, which could be
important in subsequent cultivation steps such as seedling
acclimatization.
The feasibility of using bacterial inoculants during
acclimatization, to promote plant colonization by a favor-
able rhizospheric and endophytic microbial community,
has been considered in order to optimize the development
of the plants (Khalid et al. 2004). These bacteria should
possess mechanisms for one of the following functions: (i)
biological control of phytopathogens, (ii) phytohormone
production, or (iii) supply of plant nutrients (nitrogen or
phosphate) (Christiansen-Weniger and Van Veen 1991;
Roesch et al. 2007). Bacterial inoculants can be generated
by endophytic bacteria that can colonize the rhizosphere
before penetrating host plant tissues (Azevedo 1998;
Lodewyckx et al. 2002; Andreote et al. 2006). Although
endophytes can interact with plants in different ways,
particularly important endophyte characteristics related to
plant growth promotion are the production of auxin-like
molecules (Costacurta et al. 1995; Patten and Glick 1996;
A. C. F. Dias F. D. Andreote I. S. Melo
Laboratory of Environmental Microbiology – Embrapa Meio
Ambiente, Jaguariuna, SP, Brazil
A. C. F. Dias (&) P. T. Lacava L. C. Assumpc¸a
˜
o
W. L. Arau
´
jo J. L. Azevedo
Departamento de Gene
´
tica, Escola Superior de Agricultura
‘‘Luiz de Queiroz’’, Universidade de Sa
˜
o Paulo, P.O. Box 83,
Piracicaba 13400-970, Brazil
e-mail: dias147@gmail.com
A. C. F. Dias F. E. C. Costa M. A. Teixeira
Laboratory of Microbiology, University of Vale do Sapucaı
´
–
UNIVAS, Pouso Alegre, MG, Brazil
123
World J Microbiol Biotechnol (2009) 25:189–195
DOI 10.1007/s11274-008-9878-0
Spaepen et al. 2007) and phosphate solubilization (Barea
et al. 1983; Ryan et al. 2008).
The aims of this work were firstly to identify and
characterize the capacity for phosphate solubilization and
IAA production found in endophytic bacteria that is asso-
ciated with micropropagated strawberry seedlings, and
secondly to study the ability of these strains to promote the
growth of micropropagated strawberry seedlings during the
acclimatization process.
Materials and methods
Plants used and isolation of endophytic bacteria
The bacterial samples used in the present work were
obtained from strawberry tissue culture laboratories locate
in the city of Pouso Alegre (Minas Gerais, Brazil), where
plant multiplication is performed prior to cultivation in the
field. Ten meristematic segments from each of three vari-
eties of strawberry (Camarosa, Oso-Grande and Sweet
Charlie) were selected and used for bacterial isolation.
Each plant sample consisted of a fragment containing
the meristematic region used in the micropropagation
process. Samples were surface sterilized according to a
previously described methodology for endophytic bacterial
isolation (Arau
´
jo et al. 2002). Briefly, tissues were sub-
jected to serial immersions in 70% ethanol for 1 min, 2.5%
sodium hypochlorite for 20 min, and 70% ethanol for 30 s,
followed by three rinses in sterile deionized water. The
extended time in hypochlorite (20 min) was used to
enhance the disinfection of tissues surrounding the meri-
stematic region. After surface disinfection, meristematic
regions were extracted and cut into pieces of approximately
0.2 cm which were transferred to Petri dishes containing
solid MS medium (Murashighe and Skoog 1962). The
plates were incubated at 28°C and monitored daily, over a
two week period, for bacterial colony development. After
bacterial growth had occurred, colonies were purified by
streaking, and isolated colonies were picked from the plates
and used to inoculate 5% Trypic Soy Agar slants. Colonies
were also cultured in 5% Trypic Soy Broth at 28°C for
36 h, suspended in 20% glycerol solution and stored at
-70°C.
Strain identification by FAME-MIDI
In order to identify the isolated bacterial strains, bacteria
were cultured on TSA and submitted for fatty acid methyl
ester (FAME) analysis by gas chromatography using an
automatic injector and a Flame Ionization Detector (FID)
(Agilent 6850 and 7683). The output data were organized
into a chromatogram and the identification report was
prepared using the Microbial Identification System soft-
ware (Sherlock TSBA40 library; MIDI Inc., Newark, DE,
USA). The similarity of 0.70 with hits in the database were
used to classify strains at species level, while lower values
were considered for affiliation of isolates at higher taxo-
nomic levels. The final results appeared to confirm the
similarities found between the database and the nominated
areas, enabling the strains to be identified.
Screening of endophytes for phosphate solubilization
Phosphate solubilization by the newly isolated strains was
evaluated according to the methodology previously
described by Mehta and Nautiyal (2001). Briefly, strains
were cultivated in medium PVK (glucose 1%, Ca
3
(PO
4
)
2
0.5%, (NH
4
)
2
SO
4
, 0.05%, NaCl, 0.02%, MgSO
4
7H
2
O
0.01%, KCl 0.02%, yeast extract 0.05%, MnSO
4
H
2
O,
0.0002%, FeSO
4
7H
2
O, 0.0002%, agar 1.5%), where the
ability to grow is associated to the capacity in using
Ca
3
(PO
4
)
2
as a sole phosphate source. After the cultivation
(14 days), halos were observed surrounding colonies which
were able to solubilize inorganic phosphate.
The quantitative determination of phosphate solubiliza-
tion was performed as previously described previously by
Marinetti (1962). Strains were cultivated in liquid PVK
medium, and after growing, cells were harvested (3000g
for 5 min) and supernatant were used for colorimetric
quantification of available phosphate. The staining of
phosphate in the supernatant was performed using 2 ml of
the supernatant in combination with 1 ml of vanadate
solution (NH
4
VO
3
0.25% in 35% HNO
3
) and 1 ml of
molibdate solution (NH
4
)
6
MO
7
O
24
5% in water). After the
development of a reddish color (approx. 5 min.), solutions
were submitted to spectrophotometer analysis at 420 nm.
Screening of endophytes for auxin-like molecule
production
The production of auxin molecules was determined by the
colorimetric methodology described previously by Gordon
and Weber (1951). Briefly, isolates were first screened for
IAA production by cultivation in Trypic Soy Agar plates
supplemented NH
4
Cl
2
(10 mM) and L-tryptophan
(100 lg/ml). Colonies were covered with nitrocelullose
membrane and incubated for 48 h in the dark. Membranes
were removed and stained with Salkovski reagent
(FeCl
3
6H
2
O 1.5 mM, HCl 8 M). Positive strains were
verified by the development of red staining around the
colonies. These strains were submitted to quantitative
analysis of IAA production by growing in nutrient broth
(NB) medium amended with tryptophan (100 lg/ml) in
the dark. Cells were harvested by centrifugation (12,000g
for 5 min) and the supernatant was treated with Salkovski
190 World J Microbiol Biotechnol (2009) 25:189–195
123
reagent for 15 min. The production of IAA was direct
related to the absorbance measured at 530 nm. Pure
indole-acetic-acid (IAA) was used in all experiment as a
standard.
Plant growth promotion experiment
Micropropagated strawberry seedlings of the Oso Grande
variety were used. Prior to treatment, the seedlings were
transferred to plastic tubes (9 cm high and 3.5 cm in
diameter) containing autoclaved substrate (EUCATEX,
Sa
˜
o Paulo, Brazil).
Endophytic strains used in the inoculation were grown
in liquid Nutrient Broth for 24 h at 28°C. The cells were
then harvested and bacterial cell suspensions were prepared
in water (10
8
cfu/ml
-1
). Aliquots of 100 ll from the bac-
terial suspensions were inoculated at the base of the plant
stems, near the substrate interface, in the region called the
hypocotyl. In total, 15 plants were inoculated with each
strain. Fifteen control plants received inoculation with
100 ll of water only.
Plants from all treatment groups were maintained in a
greenhouse for three months, at 30°C. The plants were then
removed from the tubes and washed in running water.
Every plant was analyzed for the following variables: root
number, length and dry weight, number of leaves, petiole
length and dry weight of shoots. The mean calculated
values for each estimated parameter were statistically
compared using standard procedures including the SAS
general linear model (GLM) and least significant difference
(LSD) analysis at the 5% level of probability (SAS Version
8.01, SAS Institute, Inc, Cary, NC).
Results and discussion
Isolation and identification of endophytic bacteria
in micropropagated strawberry plants
Endophytic bacteria colonizing in vitro strawberry tissues
were found in samples from all three varieties. However,
the frequency of endophytic isolations was found to differ
among plant genotypes. Meristematic regions of the Oso
Grande and Camarosa varieties more frequently harbor
endophytic bacteria than those of the Sweet Charlie variety
(Fig. 1). Endophytic bacteria that colonize the meriste-
matic tissues and do not cause any damage to plant
development can be surveyed for their potential to improve
plant development, conferring benefits to the plant and
resulting in an enhanced symbiotic system.
The FAME technique was used for identification and
allowed us to infer the phylogeny of 17 bacterial strains,
distributed along three genera (Bacillus, Sphingopyxis and
Virgibacillus), and comprised of two bacterial families;
Bacillaceae (Bacilli), and Sphinogmonadaceae (Alphapro-
teobacteria) (Table 1). The 20 strains were classified into
species with similarities values varying from 0.295 to
0.926 with the match found in the FAME database. FAME
identification is highly reliable for similarities higher than
0.70 at species level, while lower levels can affiliate iso-
lates to higher taxonomic groups, like genus or families
(Heyrman et al. 1999). Considering that, identified strains
were four strains of Bacillus sp., seven Bacillus subtilis,
three Bacillus megaterium, one Virgibacillus sp. and one
Sphingopyxis sp. The three remaining strains could not be
matched to any known species by the FAME technique
(Table 1). The prevalence of Bacillus spp. was postulated
to be due to possible resistance of the bacteria to the dis-
infection process prior to submission of explants to tissue
culture.
Phosphate solubilization and auxin production
The mechanisms by which the endophytic strains from
strawberry plants could influence plant growth were
investigated by assessing their capacity for phosphate sol-
ubilization and auxin production. Phosphorus, one of the
main nutrients limiting plant growth, is rapidly immobi-
lized after addition to soil as a soluble fertilizer, becoming
unavailable to the plant. Therefore, bacterial activity is
highly important with respect to supplying plants with
phosphorus. Endophytes are known to promote plant
growth by phosphate solubilization (Verma et al. 2001;
Wakelin et al. 2004). Soil inoculation with phosphate-sol-
ubilizing Bacillus spp. can solubilize fixed soil P and
applied phosphates, resulting in a better plant development
and higher yields (Yadav and Dadarwal 1997; Puente et al.
2004a and b; Canbolat et al. 2006). Qualitative phosphate
Camarosa Oso Grande Sweet Charlie
Frequency of endopytic isolation
1.0
0.0
0.2
0.4
0.6
0.8
Fig. 1 Frequency of endophytic bacteria isolation from meristematic
regions of different varieties of strawberry plants. The frequency was
determined by the number of infected samples divided by the number
of tissue fragments placed on medium plates
World J Microbiol Biotechnol (2009) 25:189–195 191
123
Table 1 Identification of bacterial strains and assessment of their growth promotion potential in strawberry seedlings
Origin Identification Seedling Growth Promotion
Roots Shoots
Variety Strains Identity Similarity (%) Number Lenght
A
(cm) Dry weight
B
(g) Number of petioles n° leaves Dry weight (g)
Camarosa C1 Bacillus megaterium 0.926 5.30 abc
A
10.65 abcd 0.039 ab 19.14 fghi 6.80 b 0.10 abc
C4 Bacillus subtilis 0.789 5.00 abc 9.72 bcde 0.025 b 21.94 cdefgh 5.91 bcd 0.10 abc
C15 Bacillus subtilis 0.863 5.91 a
B
12.08 a 0.041 ab 22.33 defgh 5.41 de 0.09 abcd
C16 Sphingopyxis sp. 0.307 5.50 abc 10.50 abcd 0.034 ab 28.77 a 7.78 a 0.13 a
C18 Bacillus sp. 0.562 5.70 ab 11.30 abc 0.041 ab 21.07 efgh 6.60 bc 0.11 ab
C19 Bacillus sp. 0.658 5.66 ab 8.66 de 0.035 ab 26.90 bcde 4.55 e 0.06 cd
C20 Bacillus sp. 0.586 5.0 abc 8.65 e 0.027 b 18.96 ghi 5.40 de 0.06 cd
C22 Not identified – 4.70 bc 12.25 a 0.023 b 17.76 fghi 5.90 bcd 0.06 d
C25 Bacillus sp. 0.295 5.5 abc 11.79 ab 0.037 ab 26.93 abcd 6.20 bcd 0.10 abc
C30 Bacillus subtilis 0.767 4.45 bc 11.50 abc 0.031 b 15.73 i 5.54 cde 0.06 cd
Oso Grande O1 Not identified – 5.92 a 12.07 a 0.045 ab 26.50 abc 6.64 bc 0.10 ab
O11 Virgibacillus sp. 0.507 5.23 abc 10.84 abc 0.042 ab 17.79 ih 5.92 bcd 0.09 abcd
O13 Bacillus megaterium 0.721 5.18 abc 10.59 abc 0.035 ab 24.22 bcdef 6.16 bcd 0.08 bcd
O17 Bacillus megaterium 0.747 4.70 bc 9.55 cde 0.028 b 28.35 ab 6.45 bcd 0.08 abcd
O19 Bacillus sp. 0.619 5.36 abc 11.45 abc 0.042 ab 19.53 fghi 5.72 bcd 0.09 abcd
O19
0
Bacillus subtilis 0.835 5.35 abc 12.32 a 0.055 a 23.21 cdefg 5.50 cde 0.11 ab
O27 Bacillus subtilis 0.913 5.54 ab 12.59 a 0.032 ab 19.56 fghi 5.33 de 0.06 cd
O28 Bacillus subtilis 0.744 5.28 abc 12.57 a 0.041 ab 25.37 abcde 6.14 bcd 0.09 abcd
O29 Bacillus subtilis 0.734 4.72 bc 12.13 a 0.039 ab 22.35 cdefgh 6.36 bcd 0.07 bcd
Sweet Charlie S8 Not identified – 4.80 bc 11.35 abc 0.036 ab 29.44 ab 5.66 bcd 0.09 abcd
Ct
C
5.10 abc 9.80 bcde 0.022 b 24.60 bcde 6.36 bcd 0.08 bcd
A
Treatments followed by a similar letter were not statistically significant (P \ 0.05)
B
Bold text indicates treatments with significantly higher values than controls (P \ 0.05)
C
Ct indicates control plants, only treated with water
192 World J Microbiol Biotechnol (2009) 25:189–195
123
solubilization activity was verified for all 20 isolates.
However, the isolates displayed variable efficiencies
(Fig. 2). Two B. subtilis strains (O19’and C4) presented the
best performance, while low indexes were observed for
strains C1 and C16, classified as B. megaterium and
Sphingopyxis sp., respectively. Although we have not done
the test for compounds involved in phosphate solubiliza-
tion, in Bacillus, the main compounds involved in the
phosphate solubilization are the lactic, itaconic, isovaleric,
isobutyric and acetic acids (Vazquez et al. 2000).
Endophytes can also promote plant growth by producing
the phytohormone IAA (Lee et al. 2004; Mendes et al.
2007). IAA increases root size and distribution, resulting in
greater nutrient absorption from the soil (Kuklinsky-Sobral
et al. 2004; Li et al. 2008). When screened for auxin pro-
duction, 15 isolates revealed to produce it at concentrations
higher than 1 lgml
-1
. Among these, the highest produc-
tion was observed by strains O13 (B. megaterium), C19
(Bacillus sp.), O29 (B. subtilis) and C16 (Sphingopyxis sp.)
(Fig. 2). High scores for phosphate solubilizers did not
match with best auxin production. For example, high pro-
duction of auxin was observed in isolate C16 (Sphingopyxis
sp.), which presented a low efficiency in solubilizing
phosphate. These data indicate that plant growth promotion
in the environment is not driven by a single species but is
due to a composite effect of features present in several
symbiotic bacteria.
Plant growth promotion by endophytic bacterial isolates
Plant growth promotion is a phenomenon driven by bene-
ficial microorganisms which associate with plants and
contribute to their better development (Christiansen-
Weniger and Van Veen 1991; Roesch et al. 2007). In this
study, plant growth promotion triggered by the inoculation
of 20 endophytic strains in strawberry seedlings under
acclimatization was evaluated by considering a number of
parameters. Inoculation of the strawberry plants with the
bacterial isolates resulted in enhanced plant development in
many cases. However, it is important to note that, in some
cases, inhibition of plant development was also observed,
indicating that the endophytic state was not always main-
tained, and may have been dependent on the cultivation
conditions.
The data from phenotypic evaluation show that from the
20 inoculated bacteria, seven promoted root growth (two
increased the number of roots, six increased the root lengths
and one increased root dry weights) and one promoted plant
shoot development (Table 1). Promotion of root develop-
ment was observed for the strains classified as B. subtilis
C15, O19, O27, O28 and O29, and for the non-identified
strain C22. In contrast, development of the aerial part of the
strawberry seedlings was promoted by strain C16, identified
as Sphingopyxis sp. (Table 1). In addition, an adaptation of
strains could be inferred, due to the better results obtained
0
5
10
15
20
C1 C4 C15 C16 C18 C19 C20 C22 C25 C30 O1 O11 O13 O17 O19 O19´ O27 O28 O29 S8 CT
25
30
35
0
5
10
15
20
25
Soluble P (microg.mL
-1
)
I
CT
C1 C4 C15 C16 C18 C19 C20 C22 C25 C30 O1 O11 O13 O17 O19 O19´ O27 O28 O29 S8 CT
CT
IAA (microg.mL
-1
)
(A)
(B)
Fig. 2 Phosphate solubilization
(a) and IAA production
(b) observed in the endophytic
bacterial strains obtained from
strawberry plants. Each value
represents the mean of three
replicates and the error bars
represent the standard
deviations of the average
World J Microbiol Biotechnol (2009) 25:189–195 193
123