Polyphasic Analysis of the Bacterial Community in the Rhizosphere and Root of
Cyperus rotundus L. Grown in a Petroleum-Contaminated Soil
Diogo Jurelevicius, Elisa Korenblum, Renata Casella, Ronalt Leite Vital and Lucy Seldin*
L b tó i de
Laboratório
d Genética
G éti Mi
Microbiana,
bi
D
Departamento
t
t dde Mi
Microbiologia
bi l i G
Geral,l IInstituto
tit t dde Mi
Microbiologia
bi l i P
Prof.f P
Paulo
l dde Gó
Góes, UFRJ
UFRJ, Rio
Ri dde JJaneiro,
i RJ
RJ.
* lseldin@micro.ufrj.br
100
INTRODUCTION
RESULTS
S [12], Rh [5]
Mycobacterium smegmatis str. MC2 155 [CP000480.1]
Mycobacterium austroafricanum strain IFP 2173 [FJ009005.1]
Mycobacterium
y
vanbaalenii PYR-1 [[CP000717.1]]
100
Petroleum represents the major energy resource
consumed throughout the industrial world (Leung et al.,
2007). As any large scale industrial process, petroleum
production can lead to contamination of soil and
groundwater (White et al., 2006), and therefore remediation
of oil pollutants is necessary. Phytoremediation, the use of
plants and their associated microorganisms to cleanup the
environmental contaminants, is an emerging technology
especially
i ll in
i the
th tropics
t i where
h
climatic
li ti conditions
diti
f
favor
plant growth and microbial activity (Gerhardt et al., 2009).
100
90
PCR-DGGE approaches
S [1]
Rh [1]
Uncultured bacterium clone SA16 [DQ182273.1]
Uncultured bacterium clone alkW1-90 [DQ288047.1]
PCR-DGGE analyses based on the 16S rRNA gene showed that the
profiles of bulk soil, rhizosphere and root samples present a high degree
of similarity (Fig.1A). A complex community of alkane utilizing-bacteria
(Fig.1B) and a variable nitrogen-fixing community (Fig.1C) were observed
when the PCR-DGGE analyses were based on the genes alkB and nifH,
respectively.
Uncultured bacterium clone OTU 21 [EU307686.1]
63
Uncultured bacterium clone alkW1-158 [DQ288065.1]
91
100
Rh [1]
Rh [1]
Uncultured bacterium clone alkW2-34 [DQ288033.1]
57
100
Uncultured bacterium clone alkW1-113 [DQ288052.1]
Rh [1]
100
60
Rh [1]
92
99
Mycobacterium avium subsp. paratuberculosis str. K10 [AE016958.1]
99
59
87
Cyperus rotundus L. is a perennial herb which was found
prevailing in an area previously contaminated with petroleum
located in the northeast of Brazil. This plant species, also
known as coco-grass, purple nut sedge or red nut sedge, is
a cosmopolitan weedy sedge that is very persistent and can
support prolonged times of drought or inundation (Pastre,
2006).) In addition,, studies have demonstrated the
involvement of C. rotundus in phytoremediation of herbicides
and heavy metals (Baodang et al., 2005).
Mycobacterium avium 104 [CP000479.1]
Mycobacterium marinum M [CP000854.1]
54
99
Rh [1]
S [4], Rh [4]
Rh [1]
Nocardia farcinica IFM 10152 [AP006618.1]
Bacterium alkW59 [DQ287999.1]
Rhodococcus erythropolis [AJ301867.1]
Pseudomonas aeruginosa PAO1 [AE004091.2]
Alcaligenaceae bacterium ITSI67 [FJ014917.1]
100
100
Enterobacteriaceae bacterium ITSI32 [FJ014916.1]
Burkholderia ambifaria MC40-6 [CP001025.1]
100
S [2], Rh [2]
Kordiimonas gwangyangensis strain S20-13 [EU853377.1]
100
Acidisphaera
p
sp.
p C197 [[AY817739.1]]
85
Uncultured bacterium clone alkb25 [EU853403.1]
98
99
0.05
59
Xanthobacter flavus [AJ418063.1]
Ralstonia sp. PT11 [DQ182291.1]
Fig. 2. Phylogenetic tree showing the affiliation of alkB clone sequences obtained from bulk and rhizosphere soil
samples (two clone libraries) to selected reference sequences. The tree was constructed based on the neighborjoining method. Bootstrap analyses were performed with 1,000 repetitions and only values higher than 50% are
shown. Scale bar represents the estimated sequence divergence. Numbers in brackets after the letters S (bulk
soil) and Rh (rhizosphere soil) correspond to the number of clones analyzed.
OBJECTIVES
To evaluate the bacterial community present in the
rhizosphere and rhizoplane/root interior of C. rotundus by
culture-dependent and molecular approaches, in order to
enlarge the knowledge of microorganism-plant interaction and
of the potential role of C. rotundus in phytoremediation.
MATERIAL & METHODS
The experimental area was located at a large on-shore petroleum
exploration and production site in Carmópolis, in the northeast of Brazil.
To study the bacterial community associated with C. rotundus, nine
plants with no significant variations in height (about 15 cm) were
harvested. These plants were grouped in three samples (A, B and C,
each constituted of three plants) and from each triplicates the
rhizosphere and rhizoplane/root samples were obtained.
i) Molecular approaches
DNA
PCR
EXTRACTION
Clone Libraries
(alkB-based)
DOTBLOTTING
ANALYSES
A total of 209 bacterial strains was isolated from the rhizosphere and
rhizoplane/roots of C. rotundus. They were selected for testing their ability for
petroleum degradation in microplates assay and the results showed that 79 isolates
were able to use crude oil as the only carbon source. Dot blotting analyses showed
that 17 oil-degrading isolates hybridized to nifH and alkB genes. The identification of
these isolates is shown in Table 1.
Fig. 1. Denaturing gradient gel electrophoresis (DGGE) fingerprints of (A) bacterial 16S rRNA, (B)
alkB and (C) nifH gene fragments amplified from bulk (S) and rhizosphere soils (Rh), and
rhizoplane/root interior (Root) of C. rotundus DNA templates; and their respective dendrograms.
Table 1. Identification by NCBI blastn (first hit) of the bacterial isolates
Isolates a Closest match
alkB-based clone libraries
Two clone libraries were generated from the alkB fragments of bulk and
rhizosphere soils. The blastn identification showed a predominance of genes
closely related to alkB from Mycobacterium species in both systems (Fig. 2).
However these two libraries were further compared with the web-LIBSHUFF
program and the results showed that the two libraries were significantly
different concerning the alkB population composition with a confidence of
95% [(Cx-Cxy)2 comparison had a p-value of 0.001].
DGGE
The 16S rRNA gene was used to study
the total bacterial community, as described
by Nübel et al. (1996). In order to access
the alkane-utilizing and the nitrogen-fixing
bacteria the genes alkB and nifH were used
as described by Chenier et al. (2003) and
Demba Diallo et al. (2004), respectively.
CONCLUSIONS
Molecular approaches showed that: (i) the total bacterial community are not
influenced by C. rotundus; and (ii) C. rotundus might select specific
functional catabolic bacteria in its rhizosphere and roots, as demonstrated
by the alkB-based
alkB based clone libraries and nifH-based
nifH based PCR-DGGE.
PCR DGGE
ii) Culture-dependent approaches
ISOLATION OF
OIL-DEGRADING
BACTERIA
Culture-Dependent Approaches
BACTERIAL
IDENTIFICATION
DOT-BLOT hybridization was performed as described by von der Weid et al. (2002).
Part of the alkB and nifH genes of strains Arh10 (isolated in this study) and
Paenibacillus durus P3L5T (from the laboratory culture collection), respectively,
generated by PCR, were used as probes.
The identification of isolates was achieved after partial sequencing of 16S rRNA
gene of each isolate.
Culture-based approaches showed that: (i) 79 isolates possess oildegrading capability; (ii) 17 isolates showed homologous sequences to alkB
and nifH genes in their genomes; (iii) These strains could be stimulating
the growth of C. rotundus in this petroleum contaminated site by the
increase of nitrogen bioavailability and/or by the degradation of petroleum
hydrocarbons, resulting in the decrease of their toxic effects.
Identity (%) Phylogenetic position b
Arh 1
Enterobacter sp. NR_024641.1
98
γ; Enterobacteriales
Aro 50
Pseudomonas sp. NR_024734.1
98
γ; Pseudomonadales
Arh 50
Mycoplana sp. NR_025831.1
98
α; Rhizobiales
Aro 1
Pandoraea sp. EU022593.1
99
β; Burkholderiales
Arh 10
Bro 5
Bro 2
Bro 1
Bro 3
Rhodococcus sp. NR_026524.1
Actinomycetales
γ; Pseudomonadales
Pseudomonas sp. DQ306309.1
98
99
99
99
99
Brh 51
Cupriavidus sp. EF189112.1
98
β; Burkholderiales
Brh 50
Cro 47
Gordonia sp. FJ459995.1
98
Actinomycetales
Cro 21
Cro 49
Rhizobium sp. AM691584.1
99
α; Rhizobiales
Cro 46
Cro 48
Bosea sp. AF273081.1
99
α; Rhizobiales
Pseudomonas sp. FJ493142.1
γ; Pseudomonadales
The isolates were named according to the samples (A, B or C, followed by ro for root and rh for rhizosphere
samples (for example: Arh 1 – sample A, rhizosphere soil, strain number). b α, Alphaproteobacteria; β,
Betaproteobacteria; γ,
γ Gammaproteobacteria.
Gammaproteobacteria
a
REFERENCES: Baodang et al. (2005) Science in China 48(1):156-164.; Chénier et al. (2003) Appl Environ Microbiol
69(9):5170-5177.; Demba Diallo et al. (2004) Environ Microbiol 6:400-415.; Gerhardt et al. (2009) Plant Sci 176:20-3.;
Leung et al. (2007) In: Modern Soil Microbiology, pp. 521-552.; Pastre (2006) IAC, Instituto Agronômico, Campinas,
SP.; Nübel et al. (1996) J Bacteriol 178:5636-5643.; von der Weid et al. (2002) Int J Syst Evol Microbiol 52:2147-2153.;
White et al. (2006) Water Air Soil Pollut 169:207-220.