Download Genetic Diversity of Rhizobium leguminosarum as Revealed

American-Eurasian J. Agric. & Environ. Sci., 13 (6): 797-801, 2013
ISSN 1818-6769
© IDOSI Publications, 2013
DOI: 10.5829/idosi.aejaes.2013.13.06.1969
Genetic Diversity of Rhizobium leguminosarum
as Revealed by 16S rRNA Gene Sequence
Mohamed Ismail, Abdelfattah M. El-Zanatay, Ragaa A. Eissa and Omar A. Hewedy
Department of Genetics, Faculty of Agriculture, Minufiya University, Shibin El-Kom, Egypt
Abstract: The genetic diversity of ten rhizobial isolates (Rhizobium leguminosarum) isolated from root nodules
of broad beans (Vicia faba L.) growing in ten locations in Egypt were investigated using 16S rRNA gene partial
sequence. The average genetic distance among the studied isolates was low (0.193) with the lowest genetic
distance between isolates collected from South Sinai and Zefta and the highest genetic distance between
North Sinai and Quesna isolates. The studied isolates formed two main groups based on cluster analyses and
principal coordinate analysis. The grouping pattern of the isolates in both analyses was independent of their
geographic location. Further, the isolation-by-distance analysis showed no correlation between genetic and
geographic distance of the studied isolates (r = 0.118, p = 0.71). The present results suggested that the genetic
diversity of Egyptian rhizobia across the studied locations is very low, probably due to the narrow genetic
background of Egyptian rhizobia.
Key words: Rhizobium leguminosarum
16S rRNA
Vicia faba
INTRODUCTION
rhizobia provides valuable bioresource for the search of
bacterial isolates in attempt to find isolates that maximize
legume crop productivity [8]. Many techniques were
developed and widely used to detect polymorphisms in
many organisms including bacteria. Among these
techniques, restriction fragment length polymorphisms
(RFLP) and direct sequence of 16S rRNA genes, which
have been used at both species and genera levels [4, 5, 9].
One of the most advantages of 16S rRNA region is
that, it is highly conservative, hence, supports the
well-established subdivision of rhizobia into species and
genera [10]. The 16S rRNA gene sequencing previously
used to detect potential novel taxa of new isolates [11].
Moreover, a longer fragment of the 16S rRNA gene
(e.g., 800 bp) contains a conserved region that is
sufficient to show the variation within groups of root
nodule bacteria [12]. So, it is anticipated to infer the
phylogenetic association among rhizobium isolates [13].
Additionally, the DNA sequence analysis of 16S rRNA
region is a powerful tool in discriminating among strains
and also known to exhibit a great deal of sequence and
length variation [14]. In the current study we examine
the genetic diversity of Rhizobium leguminosarum
sampled from cultivated soil across ten locations in Egypt.
Soil harbours different microorganisms characterized
by high level of diversity. The diversity levels within
bacteria are the highest, with approximately 50,000
bacterial species in one soil sample [1]. Rhizobia are soil
bacteria which are capable of forming nitrogen-fixing
symbiosis with different leguminous plants and have a
significant role in nutrient cycling due to biological
nitrogen fixation and enhancing crop productivity [2, 3].
These Rhizobia has an important role particularly in
low-input sustainable agriculture and land reclamation [4],
a typical situation of Egyptian agriculture. So, many of
nodule-forming bacteria are of significant agricultural
and ecological importance. The symbiosis relationships
between rhizobia and leguminous plants provide rich soil
for legumes cultivation. Many studies have addressed
the diversity level of V. faba rhizobia; mainly focusing on
rhizobial populations from the same location [5], or for
comparison with Rhizobium leguminosarum isolates from
other legume species [6]. It is important to understand the
level of genetic diversity of Rhizobium species and how
this affect soil fertility and crop productivity in response
to agricultural practices and climate [7]. The diversity of
Corresponding Author: Mohamed Ismail, Department of Genetics, Faculty of Agriculture,
Minufiya University, Shibin El-Kom, Egypt.
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Am-Euras. J. Agric. & Environ. Sci., 13 (6): 797-801, 2013
The major research questions were (i) whether the
Egyptian rhizobia isolates are originated from the same
genetic background and (ii) does close proximity
geographic locations have similar rhizobia strains?
used in PCR amplification and Big Dye Terminator v3.1
(ABI) in 3730x1 DNA analyzer (ABI) at Bioneer (Daejeon,
Korea).
Statistical Analysis
Custer Analysis: Prior to statistical analysis, the ten
sequences along with an out-group sequence (single
sequence of R. leguminosarum obtained from NCBI
database) were aligned and manually edited using BioEdit
[17]. To determine the genetic relationship among the
isolates, the number of base substitution per site among
all sequences was obtained based on the Maximum
Composite Likelihood method [18 as implemented in
MEGA4 [19]. The phylogenetic tree was reconstructed
based on Nei and Gojobori method [20] using unweighted
pair group method with arithmetic mean (UPGMA) as
implemented in MEGA4 [19]. The relative robustness of
individual branches was estimated by bootstrapping [21],
in which 1000 bootstrapped trees were generated from the
resampled data. The phylogenetic tree was then plotted
using Fig Tree 1.3.1 [22].
MATERIALS AND METHODS
Isolation of Rhizobia: A total of ten rhizobium
isolates were collected from ten fields from root
nodules on broad bean (Vicia faba L.) plants.
Rhizobium was isolated according to the methods
described by Vincent [7]. The soil samples were
collected from Quesna, Benha, Zefta, Beni Suef,
South Sinai, Suez, North Sinai, Al-Minya, Al- Dakahlia
and Cairo. These sites fall within four different agroecological zones in Egypt [15]; ranging between the Nile
as a main source of water for irrigation (Quesna, Benha,
Zefta, Beni Suef, Suez, Al-Minya and Al- Dakahlia) to
rainfed lands (South Sinaiand North Sinai). Different
broad bean verities were cultivated in these areas. Giza 3,
Misr 1, Sakha 1 and Sakha 3 are common in Nile irrigated
lands; however, Kassain and Nobaria are more common in
rainfed lands.
Principal Coordinates Analysis (PcoA): To further
determine isolates grouping, the principal coordinate
analysis (PCoA) was performed with GenAlex ver.6 [23].
This approach is complementary to cluster analysis where
the latter is more sensitive to closely related species
whereas PCoA is more prone to distances among related
groups [24].
DNA Extraction: Total genomic DNA of each isolate
was extracted from bacterial cultures grown in yeast
extract mannitol media (YEM) until late exponential phase
(109 cells mL 1). Extraction of DNA was performed using
GeneJET™ Genomic DNA Purification kit (Fermentas,
Vilnius, Lithuania) following manufacturer’s instructions.
Samples were then diluted to 20 ng DNA µL 1 and kept at
-20°C.
Isolation-by-Distance: We tested for isolation-bydistance between rhizobia isolates. This method seeks to
determine if there is a statistically significant relationship
between genetic distance and geographic distance and (2)
the strength of this relationship [25]. The correlation
between genetic and geographic distance was assessed
with Mantel test [26] with 1000 permutations as significant
level using GenAlex ver.6 [23].
Amplification of 16S rRNA Gene: The DNA of each
bacterial isolate was amplified with the universal primers,
fD1 (5' AGAGTTTGATCCTGG CTCAG 3') and rP2 (5' ACG
GCTACCTTGTTA CGACTT 3') as described in OtaTsuzuki et al. [16]. Polymerase chain reaction was
performed in 50-µL reaction volume containing 100 ng
DNA, 25 µL Maxima Hot Start PCR Master Mix
(Fermentas, Lithuania) and 20 µM of forward and reverse
primers. Amplifications were performed with the following
PCR conditions: i) initial denaturation at 95°C for 10 min,
ii) 35 cycles of 95°C for 30 s, 58°C for 1 min, 72°C for 1 min
30s and iii) 10 min final extension at 72°C.
RESULTS
Genetic Diversity of Rhizobia: Each Rhizobia isolate
produced a single fragment ranging from 1000 base pairs
(bp) to 850 bp. After sequence alignment and editing, an
average of 835 bp fragment for each isolates was
obtained. The genetic diversity was assessed as number
of base pairs substitution per site among all studied
isolates. The genetic distance among isolate pairs varied
between 0 (South Sinai and Zefta) and 0.423 (between
North Sinai and Quesna) with an average of 0.193
(Table 1).
Partial 16S rRNA Gene Sequencing: Based on the PCR
results, all ten isolates showed a single fragment
amplification which were subsequently used for partial
sequencing of the 16S rRNA gene. The 16S rRNA genes
were sequenced from both strands using the same primers
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Am-Euras. J. Agric. & Environ. Sci., 13 (6): 797-801, 2013
Table 1: Number of base substitution per site between 10 Rhizobium legumizarum isolates using Nei and Giobori method
Quesna
Benha
Zefta
Beni Suef
South Sinai
Suez
North Sinai
Al-Minya
Al-Dakahlia
Cairo
Quesna
Benha
Zefta
Beni Suef
South Sinai
Suez
North Sinai
Al-Minya
Al-Dakahlia
Cairo
0.000
0.258
0.26
0.029
0.259
0.290
0.423
0.234
0.019
0.058
0.000
0.163
0.211
0.163
0.188
0.298
0.152
0.223
0.216
0.000
0.217
0.000
0.034
0.305
0.05
0.229
0.211
0.000
0.216
0.246
0.366
0.193
0.009
0.022
0.000
0.034
0.304
0.050
0.228
0.21
0.000
0.303
0.069
0.259
0.247
0.000
0.273
0.381
0.371
0.000
0.204
0.195
0.000
0.033
0.000
Fig. 1: Phylogenetic relationship of 10 Rhizobium legumizarum isolates using UPGMA method. Number next to the
branches represents bootstrap values
Fig. 2: Two-dimensional representation of genetic relationships among 10 Rhizobium leguminosarum isolates
determined on the basis of principal coordinates analysis of 16S rRNA gene sequence according to the first two
principal coordinates (PC1 and PC2)
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Am-Euras. J. Agric. & Environ. Sci., 13 (6): 797-801, 2013
analysis is presented in Fig. 1 supported this hypothesis
where the groups did not correspond to geographic
location. Similar pattern was previously reported by Yang
and Zhou [27] where they found that rhizobia population
in China probably originated from those of Japan and
North America.
The phylogenetic relationship (Fig. 2) showed that
isolates from lower delta locations (Cairo, Quesna and
Al-Dakahlia) formed one group in addition to isolate
from Beni Suef which is about 224 kilometer (Km) from
Al-Dakahlia. On the other hand, the grouping pattern of
the remaining isolates was rather puzzling. Isolates from
Zefta and South Sinai formed one group with 100%
bootstrap support although they are located more than
400 km apart. Remarkably, isolates from North and South
Sinai came in one group although they did not form an
adjacent group. It is well-known that the highly conserved
nature of the 16S rRNA gene sequence, the existence of
similar genotypes or similar sequences in distantly
separated location is expected under varying
environmental conditions [6].
To further investigate grouping pattern of the
studied rhizobia, we applied PCoA as another tool to
assess such pattern from different prospective. The first
and second PCoA axis explained 75% of the variation
within the sequence data (Fig. 2) which represent
reasonable coverage of the total variation. These results
showed a stronger, yet similar, pattern of grouping
compared to cluster analysis. However, the grouping
pattern was irrespective to the isolates geographic
locations. To assess the relationship between genetic and
geographic an isolation-by-distance test was used to
decipher if the genetic distance independent of
geographic distance. As expected, no correlation between
genetic distance and geographic distance was observed,
supporting the notion of single origin of Egyptian
rhizobia. The presence of recombination within and
between ribosomal genes has been documented [12],
which might be involved in the Egyptian rhizobia genetic
homogeneity.
Fig. 3: Correlation between genetic distance (base
substitution per site) and geographic distance
(km) of 10 Rhizobium legumizarum isolates.
Cluster analysis revealed two main clusters
(Figure 1).
The first cluster included isolates from Zefta, South
Sinai, Suez, Al-Minya, Benha and North Sinai; whereas
the second cluster included isolates from Cairo, Beni Suef,
Quesna and Al- Dakahlia. Principal coordinates analysis
showed similar pattern as cluster analysis. The first and
second principal coordinates explained 50.14% and
26.64% of the variation within isolates, respectively
(Fig. 2).
The studied isolates formed two distinct groups,
the first group included isolates from Zefta, South Sinai,
Suez, Al-Minya, Benha and North Sinai; whereas, the
second group included isolates from Cairo, Beni Suef,
Quesna and Al- Dakahlia. Remarkably, the isolates within
each group were overlapped except North Sinain and
Benha isolates were distinct from other isolates in the
second group (Fig. 2). The results of mantel test indicated
a week correlation between genetic and geographical
distance (Fig. 3, r = 0.118, p = 0.71).
DISCUSSION
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