RESEARCH LETTER
Culturable bacterial community analysis in the root domains of two
varieties of tree peony (Paeonia ostii )
Jigang Han1, Yao Song1, Zhigang Liu1,2 & Yonghong Hu1
1
Shanghai Engineering Research Center of Sustainable Plant Innovation, Shanghai Botanical Garden, Shanghai, China; and 2College of Agriculture,
Henan University of Science and Technology, Luoyang, China
Correspondence: Yonghong Hu, Shanghai
Engineering Research Center of Sustainable
Plant Innovation, Shanghai Botanical Garden,
1111, Longwu Road, Shanghai, 200231,
China. Tel./Fax.: 186-21-54363368;
e-mail: [email protected]
Received 12 April 2011; revised 20 May 2011;
accepted 22 May 2011.
Final version published online 20 July 2011.
DOI:10.1111/j.1574-6968.2011.02319.x
Editor: Yaacov Okon
MICROBIOLOGY LETTERS
Keywords
culturable bacteria; amplified ribosomal DNA
restriction analysis; tree peony (Paeonia ostii);
diversity.
Abstract
A total of 985 bacterial strains with different colony characteristics were isolated
from the root of tree peony plants (variety ‘Fengdan’ and ‘Lan Furong’); 69
operational taxonomic units were identified by amplified ribosomal DNA restriction analysis. Representatives of each group were selected for partial 16S rRNA
gene sequencing and phylogenetic analysis. The major groups in the bulk soil,
rhizosphere, and rhizoplane of Fengdan were Firmicutes (63.2%), Actinobacteria
(36.3%), and Betaproteobacteria (53.0%), respectively. The major bacteria groups
in the bulk soil, rhizosphere, and rhizoplane of Lan Furong were Actinobacteria
(34.8%), Gammaproteobacteria (45.2%), and Betaproteobacteria (49.1%), respectively. In total, the bacterial isolates comprised 26 genera – 14 in the bulk soil, 14 in
the rhizosphere, and 11 in the rhizoplane. The most common genus in the bulk soil
of Fengdan and Lan Furong was Bacillus (49.6% and 32.6%, respectively), in the
rhizosphere Microbacterium (21.1%) and Pseudomonas (42.0%), and in the rhizoplane Variovorax (53.0% and 49.1%, respectively). The results show that there are
obvious differences in the bacterial communities in the three root domains of the
two varieties, and the plants exerted selective pressures on their associated bacterial
populations. The host genotypes also influenced the distribution pattern of the
bacterial community.
Introduction
Plant-associated bacteria (PAB) reside in the rhizosphere,
phyllosphere, and tissues of healthy plants, and have diverse
abilities to affect plant health, their genotypic and phenotypic characteristics, and their phylogeny (Beattie, 2006).
PAB are part of the natural microbial communities of
healthy plants and it is clear that many plant-associated
microorganisms, even those that constitute only a small
proportion of a community, can have functions that are of
agricultural or environmental importance, especially as
agents for stimulating plant growth and managing soil
(Hallmann et al., 1997; Compant et al., 2005; Han et al.,
2005), designated as plant growth-promoting bacteria
(PGPB). Bacterial mechanisms of plant growth promotion
include biological nitrogen fixation, synthesis of phytohormones, environmental stress relief, synergism with other
bacteria–plant interactions, inhibition of plant ethylene
synthesis, as well as increasing availability of nutrients such
FEMS Microbiol Lett 322 (2011) 15–24
as phosphorus, iron and minor elements, and growth
enhancement by volatile compounds (Fuentes-Ramirez &
Caballero-Mellado, 2005).
Technical advances in microbial ecology and genomics
have been paralleled by advances in our understanding of
the structure and dynamics of these plant-associated microbial communities and the molecular basis of plant–microorganism and microorganism–microorganism interactions.
A large body of literature has described the crop plantassociated bacterial community and its applications in
agriculture, and some strains have been developed as
biofertilizers (Podile & Kishore, 2006). However, little
research has focused on the ornamental plant-associated
bacterial community and its applications.
PAB have been isolated from many crop plant species
(Rosenblueth & Martinez-Romero, 2006), including rice
(Engelhard et al., 2000), soybean (Kuklinsky-Sobral et al.,
2004), potato (Asis & Adachi, 2004), wheat (Coombs &
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16
Franco, 2003) and maize (Zinniel et al., 2002), as well as
ornamental plants, such as tulsi (Tiwari et al., 2010),
avocado (Cazorla et al., 2007), and palm (Rivas et al.,
2007). There is a great opportunity to find new and
interesting plant-associated microorganisms among the
myriads of plants in different settings and ecosystems. Until
now, relatively little information has been available regarding the bacterial community associated with the tree peony.
The tree peony is an important ornamental plant indigenous to China, belonging to the section Moutan in the
genus Paeonia, Paeoniaceae. In China, the tree peony has
been cultivated since the Dongjin Dynasty 1600 years ago
and it was introduced to Japan early in 724–749 and brought
to Europe in 1787 (Li, 1999). The root bark of the tree
peony, known as Dan Pi, is an important ingredient in
Chinese traditional medicine (Pan & Dai, 2009; Li et al.,
2010). All wild species are widely dispersed in China, and
more than 1500 cultivars have been planted (Han et al.,
2008). In spite of this diversity, many cultivars with
good ornamental traits do not grow well in some areas
because of the poor soil and climate conditions. For
example, some Zhongyuan and Xibei cultivars such as Lan
Furong do not grow well south of the Yangtze River in
China. A good way to screen for and apply PGPB strains to
tree peony cultivation might be based on the characteristics
of PGPB strains. We therefore investigated the application
of the PGPB strains of the plant-associated bacterial
community.
In this study, bacteria were isolated from the bulk soil,
rhizosphere, and rhizoplane in the root of tree peony plants
collected from Luoyang, China. The diversity of culturable
bacteria was investigated by amplified ribosomal DNA
restriction analysis (ARDRA) and 16S rRNA gene sequence
analysis. To the best of our knowledge, this is the first report
of PAB diversity of tree peony plants.
Materials and methods
Soil samples
Soil samples were obtained from Luoyang National Peony
Garden (Luoyang, Henan Province, China), where different
varieties were cultured in different sections. Sampling was
conducted according to the methods described by Han et al.
(2009) with some modifications. In November 2009, rhizosphere and rhizoplane soil samples from the root domain of
tree peony (Paeonia ostii) of two varieties (Fengdan and Lan
Furong), each of three plants, representing about 10-year
growth, were collected randomly at a depth of 5–15 cm from
the stem base, with each plant at least 50 m from each other.
Bulk soil samples were collected according to the previous
methods at the same time. Samples were analyzed for
recovery of isolates 8–10 h after collection.
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J. Han et al.
Isolation of bacteria, media, and culture
conditions
Rhizosphere, rhizoplane, and soil bacteria were isolated
according to the previous procedures (Courchesne &
Gobran, 1997; Han et al., 2005) with Luria–Bertani (LB,
1 , and 0.1 ), trypticase soy agar (TSA), yeast–glucose
(YG), R2A, and King’s B (KB) plates. In all cultivation
experiments, the agar plates were incubated in the dark for
3–5 days at 28 1C. Based on the colony characteristics, single
colonies with different morphological characteristics were
selected and stored in 15% glycerol at 80 1C for further
study.
DNA extraction, PCR amplification, and ARDRA
analysis
The DNA of bacterial isolates was prepared according to the
procedures of Park et al. (2005). The 16S rRNA genes were
amplified from genomic DNA by PCR using the primers 27F
and 1378R (Weber et al., 2001). The 50 mL PCR mixture
contained 20–50 ng DNA, 250 pmol of each primer, 5 mL
10 PCR buffer, 2.5 U rTaq DNA polymerase (TaKaRa,
Shanghai), and 100 mM dNTP mixture. The PCR program
consisted of a denaturation step of 94 1C for 5 min and 30
cycles of 94 1C for 1 min, 48 1C for 45 s, and 72 1C for 45 s,
followed by a final extension step at 72 1C for 10 min.
Amplification products were examined by agarose gel electrophoresis and purified using the PCR Purification Kit
(Gold Chain BioTech Centre, Beijing) according to the
manufacturer’s protocol.
ARDRA was performed to group the isolates into different phylotypes. 16S rRNA gene was digested using the four
base-cutting restriction enzymes MspI and AluI (1 U) at
37 1C for 1 h (Costa et al., 2006). The restricted products
were electrophoresed in 2.5% agarose gel and the patterns in
the gels were compared. Representative phylotypes were
sequenced with the primers 968F-1492R and 27F-1378R
(Weber et al., 2001) on an ABI 3100 DNA sequencer by
the Chinese National Human Genome Center (Shanghai,
China).
Calculation of diversity indices
An operational taxonomic unit (OTU) was defined as a 16S
rRNA gene digestion group in a same profile in ARDRA.
Phylotype richness (S) was calculated as the total number
of OTUs. The Shannon–Wiener index was calculated as
follows:
H¼
S
X
Pi ln Pi
i¼1
where Pi is the frequency of the ith type in the population
(Martin, 2002).
FEMS Microbiol Lett 322 (2011) 15–24
17
Culturable bacterial community in the root of tree peony
Phylogenetic analysis
The presence of possible chimeric sequences was investigated using the CHIMERA-CHECK program of the Ribosomal
Database Project II (Maidak et al., 1999). The most similar
sequences were searched within the NCBI database (http://
www.ncbi.nlm.nih.gov/) using the BASIC LOCAL ALIGNMENT
SEARCH TOOL (BLAST) and the sequences obtained in this study
were deposited in GenBank (for the accession numbers, see
Tables 1 and 2).
Results
Quantification of bacterial populations
A total of 985 bacterial strains with different colony characteristics were isolated on LB, TSA, YG, R2A, 0.1 LB, and
KB media: 349 isolates from the rhizosphere, 172 isolates
from rhizoplane, and 464 isolates from the bulk soil of the
two tree peony varieties plants (Fengdan and Lan Furong),
respectively (Table 3). The highest bacterial numbers of the
Fengdan and Lan Furong plants were, respectively,
3.14 107 YG and 8.94 107 R2A CFU g1 of bulk soil,
1.17 108 R2A and 2.31 108 R2A CFU g1 of fresh root for
the rhizosphere, 2.83 107 R2A and 5.73 107 YG CFU g1
of fresh root for the rhizoplane. R2A plate has the highest
number of isolates for most of the samples, except Lan
Furong rhizoplane and Fengdan bulk soil samples, whereas
LB plate has the lowest number of isolates for most of the
samples, except Fengdan rhizosphere and rhizoplane soil
samples. On the different plates, the bacterial population
density of the Lan Furong rhizosphere is 1.5–2.0 times that
of Fengdan, and the density of the Lan Furong rhizoplane
1.4–5.7 times that of Fengdan.
ARDRA analysis and bacterial diversity of the
bulk soil, rhizosphere, and rhizoplane in the root
domain of tree peony plants
In all, 507 isolates obtained from Fengdan samples and 478
isolates obtained from Lan Furong samples were subjected
to ARDRA analysis by digestion of the amplified 16S rRNA
gene with MspI and AluI. The same banding patterns
obtained after the double digestions were grouped and
defined as an OTU. A total of 161 isolates and 188 isolates
from rhizosphere of Fengdan and Lan Furong were grouped
into 21 OTUs and 20 OTUs; 66 isolates and 106 isolates
from rhizoplane of Fengdan and Lan Furong were grouped
into nine OTUs and 10 OTUs; and 280 isolates and 184
isolates from the bulk soil of Fengdan and Lan Furong were
grouped into 18 OTUs and 10 OTUs, respectively (Table 3).
In all the cases, the largest number of OTUs (48) were
obtained from R2A plates, in contrast to 28 OTUs from LB
FEMS Microbiol Lett 322 (2011) 15–24
plates, the smallest number (Table 3). R2A is therefore the
optimal media to isolate bacterial strains in the root
domains of tree peony plants.
The phylotypes using the Shannon–Wiener index (H) of
the bacterial communities in the bulk soil, rhizosphere, and
rhizoplane of Fengdan and Lan Furong were calculated –
2.41, 2.71, 1.87 and 2.1, 2.38, 1.69, respectively.
Phylogenetic analysis of bacterial communities
Representatives of each group were selected for partial 16S
rRNA gene sequencing to retrieve sequence similarity and
bacterial identity from sequence databases. All of the bacterial isolates from Fengdan and Lan Furong were assigned to
five phyla within the domain Bacteria, namely Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria (Tables 1, 2, and 4).
The bulk soil isolates from Fengdan and Lan Furong were
represented by three phyla and five phyla: Firmicutes
(63.2%), Betaproteobacteria (17.2%), Actinobacteria (19.6%),
and Firmicutes (32.6%), Alphaproteobacteria (0.5%), Betaproteobacteria (8.7%), Gammaproteobacteria (23.4%), and
Actinobacteria (34.8%), respectively. Bacterial isolates from
bulk soil of Fengdan and Lan Furong were assigned to nine
and eight genera, respectively. The genus Bacillus was the
major taxon in both of the bulk soil samples of Fengdan and
Lan Furong (49.6% and 32.6%, respectively) (Tables 1, 2,
and 4).
The rhizosphere isolates from both Fengdan and Lan
Furong were represented by five phyla. The majority of the
isolates from rhizosphere soil of Fengdan were in the
Actinobacteria group (36.3%), whereas the most abundant
group in the rhizosphere soil of Lan Furong was the phylum
Gammaproteobacteria (45.2%). Ten genera were found in
rhizosphere bacterial isolates from Fengdan and Lan Furong, respectively. Microbacterium (21.1% and 11.7%), Bacillus (15.5% and 18.1%), Variovorax (18.6% and 20.7%),
and Pseudomonas (16.8% and 42.0%) represented 72% and
92.5% of the isolates from the rhizosphere of Fengdan and
Lan Furong plants, respectively (Tables 1, 2, and 4).
The phylogenetic analysis indicated that the isolates from
the rhizoplane of Fengdan and Lan Furong could also be
grouped into four phyla: Betaproteobacteria (53.0% and
49.1%), Actinobacteria (19.7% and 16.7%), Alphaproteobacteria (16.7% and 17.0%), and Gammaproteobacteria (10.6%
and 17.0), respectively. Eight and seven genera were identified in the rhizoplane bacterial isolates from Fengdan and
Lan Furong, respectively. Members of Betaproteobacteria
were predominant (53.0% and 49.1%) in both varieties,
and all isolates in this group were Variovorax, which also was
the major genera (Tables 1, 2, and 4).
In total, the bacterial isolates comprised 26 genera – 14 in the
bulk soil, 14 in the rhizosphere, and 11 in the rhizoplane roots.
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c
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Gammaproteobacteria
Betaproteobacteria
Alphaproteobacteria
Agromyces
Actinobacteria
Xanthomonas
Pseudomonas
Lysobacter
Variovorax
Sphingobium
Sinorhizobium
Cupriavidus
Mycobacterium
Nocardioides
Streptomyces
Agrobacterium
Bosea
Ensifer
Nitratireductor
Phenylobacterium
Sphingopyxis
Agrococcus
Cellulosimicrobium
Microbacterium
Arthrobacter
Genus
Phylogenetic group
A. aruantiacus
A. allii
A. italicus
A. pascens
A. sulfonivorans
A. jenensis
C. cellulans
M. arborescens
M. insulae
M. thalassium
M. hominis
M. trichotecenolyticu
M. flavescens
M. phyllosphaerae
M. neoaurum
N. hankookensis
S. gulbargensis
A. tumefaciens
B. eneae
E. adhaerens
N. basaltis
P. koreense
S. ginsengisoli
S. witflariensis
S. estrogenivorans
S. meliloti
C. respiraculi
C. taiwanensis
V. koreensis
V. soli
V. paradoxus
L. gummosus
L. daejeonensis
L. niabensis
P. brassicacearum
P. chlororaphis
P. fluorescens
P. migulae
P. putida
P. resinovorans
Pseudomonas sp.
P. thivervalensis
X. campestris
Species
48
14
3
10
1
6
10
11
Fengdan (280)
Bulk soil
28
15
16
1
16
3
16
29
Lan Furong (184)
18
8
1
6
30
1
5
21
5
8
18
8
Fengdan (161)
Rhizosphere
61
8
6
1
3
36
3
6
1
3
22
4
Lan Furong (188)
Table 1. Distribution of representative bacterial taxa in the bulk soil, rhizosphere, and rhizoplane of tree peony plants (‘Fengdan’ and ‘Lan Furong’)
7
15
20
5
1
5
2
9
2
Fengdan (66)
Rhizoplane
15
3
50
2
3
6
9
9
9
Lan Furong (106)
18
J. Han et al.
FEMS Microbiol Lett 322 (2011) 15–24
FEMS Microbiol Lett 322 (2011) 15–24
Bacillus
Firmicutes
c
B. bataviensis
B. cereus
B. drentensis
B. firmus
B. horneckiae
B. indicus
B. megaterium
B. niabensis
B. niacini
B. pumilus
B. psychrodurans
B. pichinotyi
B. soli
Bacillus sp.
P. pabuli
S. psychrophila
S. soli
S. epidermidis
T. saccharophilus
Species
32
6
14
9
34
4
1
5
63
9
Fengdan (280)
Bulk soil
35
25
Lan Furong (184)
Numbers indicate strains assigned to each species. Numbers in parentheses are the total numbers of isolates.
Staphylococcus
Terribacillus
Paenibacillus
Sporosarcina
Genus
Phylogenetic group
Table 1. Continued.
2
5
1
1
6
3
8
6
Fengdan (161)
Rhizosphere
1
3
19
6
2
3
Lan Furong (188)
Fengdan (66)
Rhizoplane
Lan Furong (106)
Culturable bacterial community in the root of tree peony
19
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J. Han et al.
Table 2. List of isolates representing the 48 OTUs generated by ARDRA analysis from bulk soil, rhizosphere, and rhizoplane of tree peony plants
(‘Fengdan’)
Root
domains
Phylogenetic group
(genus)
ARDRA
group
Representative isolate (accession
no.)
Bulk soil
Agromyces
Arthrobacter
Bacillus
LF1
LF2
LF3
LF4
LF5
LF6
LF7
LF8
LF9
LF10
LF11
LF12
LF13
LF14
LF15
LF16
LF17
LF18
LF19
LF20
LF21
LF22
LF23
LF24
LF25
LF26
LF27
LF28
LF29
LF30
LF31
LFS13 (JF682057)
LFS15 (JF682059)
LFS17 (JF682061)
LFS84 (JF682072)
LFS18 (JF682062)
LFS22 (JF682063)
LFS23 (JF682064)
LFS80 (JF682070)
LFS24 (JF682065)
LFS81 (JF682071)
LFS88 (JF682073)
LFS44 (JF682067)
LFS16 (JF682060)
LFS95 (JF682074)
LFS14 (JF682058)
LFS64-1 (JF682069)
LFS53 (JF682068)
LFS30 (JF682066)
LFR6-1 (JF682038)
LFR30 (JF682044)
LFR15 (JF682052)
LFR43 (JF682055)
LFR39 (JF682053)
LFR41 (JF682054)
LFR6-2 (JF682051)
LFR67 (JF682056)
LFR1 (JF682037)
LFR10 (JF682040)
LFR11 (JF682041)
LFR22 (JF682043)
LFR19 (JF682042)
LF32
LF33
LF34
LF35
LF36
LF37
LF38
LF39
LF40
LF41
LF42
LF43
LF44
LFR7 (JF682039)
LFR75 (JF682048)
LFR35 (JF682047)
LFR3 (JF682049)
LFR4 (JF682050)
LFR68 (JF682045)
LFR21 (JF682046)
LFP9 (JF682031)
LFP22 (JF682035)
LFP24 (JF682036)
LFP16 (JF682033)
LFP15 (JF682032)
LFP1 (JF682028)
LF45
LF46
LF47
LFP5 (JF682030)
LFP2 (JF682029)
LFP20 (JF682034
Cupriavidus
Microbacterium
Nocardioides
Paenibacillus
Streptomyces
Terribacillus
Rhizosphere Agromyces
Arthrobacter
Bacillus
Cupriavidus
Lysobacter
Microbacterium
Pseudomonas
Sporosarcina
Sphingopyxis
Variovorax
Rhizoplane Agromyces
Agrobacterium
Microbacterium
Mycobacterium
Sphingopyxis
Sphingobium
Variovorax
Xanthomonas
Although isolates of Agromyces, Microbacterium, Variovorax,
and Lysobacter were found in all three root domains,
many isolates were found only in a single domain. For example,
strains of Agrococcus, Streptomyces, Nocardioides, Ensifer, Paeni2011 Federation of European Microbiological Societies
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Nearest type strain (accession no.)
Agromyces aruantiacu (DQ870725)
Arthrobacter sulfonivorans (FM955860)
Bacillus drentensis (NR_029002)
Bacillus firmus (AJ717383)
Bacillus horneckiae (EU861362)
Bacillus indicus (NR_029022)
Bacillus megaterium (EU124555)
Bacillus niabensis (AB271138)
Bacillus pumilus (FJ032017)
Bacillus psychrodurans (AM086235)
Cupriavidus respiraculi (EU827493)
Microbacterium arborescens (DQ870704)
Microbacterium insulae (EU239498)
Microbacterium thalassium (AM181507)
Nocardioides hankookensis (EF555584)
Paenibacillus pabuli (AM087615)
Streptomyces gulbargensis (DQ317411)
Terribacillus saccharophilus (AB243845)
Agromyces italicus (AY618215)
Arthrobacter pascens (AJ576068)
Bacillus bataviensis (AJ542508)
Bacillus cereus (GQ462533)
Bacillus niabensis (AB271138)
Bacillus niacini (NR_024695)
Bacillus pichinotyi (FJ639186)
Bacillus soli (AJ542514)
Cupriavidus taiwanensis (NR_028800)
Lysobacter gummosus (FN313519)
Microbacterium hominis (AM181504)
Microbacterium insulae (EU239498)
Microbacterium trichotecenolyticum
(Y17240)
Pseudomonas chlororaphis (EU834423)
Pseudomonas migulae (NR_024927)
Pseudomonas resinovorans (EU019983)
Sporosarcina psychrophila (D16277)
Sporosarcina soli (DQ073394)
Sphingopyxis ginsengisoli (AB245343)
Variovorax soli (DQ432053)
Agromyces allii (DQ673874)
Agrobacterium tumefaciens (FJ666055)
Microbacterium insulae (EU239498)
Mycobacterium neoaurum (FJ172307)
Sphingopyxis witflariensis (NR_028010)
Sphingobium estrogenivorans
(DQ855413)
Variovorax koreensis (EU827487)
Variovorax paradoxus (GQ221846)
Xanthomonas campestris pv. (EU364838)
Sequence identity
(%)
98
99
99
97
97
98
98
97
97
97
97
98
98
98
98
98
98
99
95
97
98
97
97
96
98
96
98
99
98
98
99
99
98
98
96
99
97
97
98
99
98
99
98
97
98
99
94
bacillus, and Terribacillus were only found in the bulk soil;
strains of Sporosarcina, Lysobacter, Cellulosimicrobium, Bosea,
Nitratireductor, and Staphylococcus only in the rhizosphere, and
strains of Xanthomonas, Agrobacterium, Mycobacterium,
FEMS Microbiol Lett 322 (2011) 15–24
21
Culturable bacterial community in the root of tree peony
Table 3. Quantification and OTUs of bacterial populations in the root domain of ‘Fengdan’ and ‘Lan Furong’ with different media
Bulk soil
Rhizospheric soil
Fengdan
Lan Furong
Fengdan
Rhizoplane soil
Lan Furong
Fengdan
Lan Furong
Media
w
w
w
w
w
w
Total
OTUs
R2A
LB
0.1 LB
YG
KB
TSA
24.8
8.9
15.0
31.4
12.4
9.74
15
8
10
13
9
9
89.4
38.0
59.2
69.9
61.0
44.4
9
6
7
8
7
5
117.0
43.6
64.4
113.0
73.6
42.6
18
11
11
20
9
11
231.0
63.4
123.0
156.0
115.0
75.7
18
13
11
15
11
8
28.3
4.4
18.6
28.1
9.9
2.2
6
4
5
5
4
4
40.5
7.7
47.4
57.3
35.5
12.6
8
5
5
7
4
5
48
28
32
43
35
29
Quantification of bacterial populations ( 106 CFU g1).
w
OTUs of bacterial populations.
Phenylobacterium, Sphingobium, and Sinorhizobium only in
the rhizoplane (Tables 1, 2, and 4).
Discussion
We noticed that the population density of culturable rhizobacteria was higher than that of bulk soil and rhizoplane
bacteria, regardless of the media plate used and the variety.
These results are similar to previous reports (Li et al., 2008).
The root surrounding rhizosphere contains compounds
such as free amino acids, proteins, carbohydrates, alcohols,
vitamins, and hormones which are important sources of
nutrients for the microorganisms present in the rhizosphere
and attract a great diversity and population density of
microorganisms (Compant et al., 2005; Han et al., 2005).
This distribution pattern confirms and extends results
reported previously for sugarcane (Mendes et al., 2007),
maize, and coffee plants (Estrada-De et al., 2001). However,
the incubation time of 3–5 days was too short to reveal those
slow-growing bacteria, and further work with longer incubation times is needed to overcome this bias.
There were obvious differences among the bulk soil,
rhizosphere, and rhizoplane bacterial communities in the
root domain of the two peony varieties Fengdan and Lan
Furong. The main differences in the bacterial community
structure occurred in the bulk soil of the two varieties, which
was represented by three and four phyla, respectively. Also,
only two genera, Microbacterium and Bacillus, were found
together in the bulk soil of the two varieties, although the
members of the genus Bacillus were the major taxon in both
of the bulk soil samples of Fengdan and Lan Furong.
Aside from the differences in the bacterial community
structure, the bacterial population density in bulk soil of the
two varieties was also different; the density of Lan Furong
was 2.2–4.9 times that of Fengdan on the different plates. It
is possible that this is a result of different culture methods
for these two varieties plants. The Lan Furong plants were
given much more fertilizer and cultivation because the
ornamental traits of Lan Furong are much better than those
FEMS Microbiol Lett 322 (2011) 15–24
of Fengdan. Usually, Fengdan plants are cultured only as a
stock for grafting in Luoyang National Peony Garden.
The bacterial community structure in the bulk soil was
similar to that of the rhizosphere in the two varieties of
peony. The same five phyla were found in the rhizosphere
bacterial community structure, although the predominant
phylum was different. The Actinobacteria group was the
most abundant in the rhizosphere of Fengdan plants,
compared with the Gammaproteobacteria group in the
rhizosphere of Lan Furong plants (Tables 1, 2, and 4;
Fig. 1). Among 14 genera in the rhizosphere of the two
varieties plant, five genera (Microbacterium, Variovorax,
Lysobacter, Sporosarcina, and Bacillus) were found at the
same time. The bacterial community structure in the rhizoplane of the two varieties was much more similar than the
other two domains in the root of the plants. Both were
represented by four phyla with similar percentages. The
predominant phylum was also same as Betaproteobacteria.
Moreover, members of Bacillus and Pseudomonas were
absent at the same time in the rhizoplane of the two varieties
of peony. It would appear that a selective pressure of tree
peony plants on their associated bacterial populations
occurred, as has been observed before (Lilley et al., 1996;
Hallmann et al., 1997). The maximum effects have been seen
near the root surface because both are distinct ecological
niches where specific nutritional selection occurs (Marilley
& Aragno, 1999; Siciliano & Germida, 1999; Jung et al.,
2008).
Many isolates were found only in the root of Fengdan or
Lan Furong in this study. For example, strains of Agromyces,
Arthrobacter, Sphingopyxis, and Cupriavidus were only found
in the rhizosphere of Fengdan, and strains of Cellulosimicrobium, Bosea, Ensifer, and Staphylococcus were only found in
rhizosphere of Lan Furong. Strains of Agromyces, Mycobacterium, Sphingopysix, and Sphingobium were only found in
the rhizoplane of Fengdan, compared with strains of Phenylobacterium, Sinorhizobium, and Lysobacter in the rhizoplane
of Lan Furong. As the bacterial population densities in the
rhizosphere and rhizoplane of Lan Furong were both higher
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
22
J. Han et al.
Table 4. List of isolates representing the 39 OTUs generated by ARDRA analysis from bulk soil, rhizosphere, and rhizoplane of tree peony plants (‘Lan
Furong’)
Root
domains
Phylogenetic group
(genus)
ARDRA
group
Representative isolate (accession
no.)
Bulk soil
Arthrobacter
Agrococcus
Bacillus
LL1
LL2
LL3
LL4
LL5
LL6
LL7
LL8
LL9
LLS1 (JF682102)
LLS8 (JF682103)
LLS21 (JF682105)
LLS33 (JF682108)
LLS12 (JF682104)
LLS32 (JF682107)
LLS24 (JF682106)
LLS37 (JF682109)
LLS59 (JF682111)
LL10
LL11
LL12
LL13
LL14
LL15
LL16
LL17
LL18
LL19
LL20
LL21
LL22
LL23
LL24
LL25
LL26
LL27
LL28
LL29
LL30
LL31
LLS44 (JF682110)
LLR38 (JF682095)
LLR37 (JF682094)
LLR27 (JF682089)
LLR32 (JF682092)
LLR21 (JF682088)
LLR19 (JF682085)
LLR18 (JF682086)
LLR84 (JF682100)
LLR1 (JF682084)
LLR48 (JF682096)
LLR61 (JF682097)
LLR28 (JF682090)
LLR30 (JF682091)
LLR34 (JF682093)
LLR20 (JF682087)
LLR63 (JF682098)
LLR96 (JF682101)
LLR74 (JF682099)
LLP32 (JF682083)
LLP30 (JF682082)
LLP2 (JF682075)
LL32
LL33
LL34
LL35
LL36
LL37
LLP5 (JF682077)
LLP20 (JF682081)
LLP7 (JF682078)
LLP16 (JF682080)
LLP3 (JF682076)
LLP8 (JF682079)
Ensifer
Lysobacter
Microbacterium
Pseudomonas
Variovorax
Rhizosphere Bacillus
Bosea
Cellulosimicrobium
Lysobacter
Microbacterium
Nitratireductor
Pseudomonas
Sporosarcina
Staphylococcus
Variovorax
Rhizoplane Agrobacterium
Lysobacter
Microbacterium
Phenylobacterium
Sinorhizobium
Variovorax
Xanthomonas
Nearest type strain (accession no.)
Sequence identity
(%)
Arthrobacter pascens (AJ576068)
98
Agrococcus jenensis (AJ717350)
98
Bacillus firmus (AJ717383)
98
Bacillus drentensis (NR_029002)
97
Ensifer adhaerens (AB513651)
99
Lysobacter daejeonensis (DQ191178)
98
Microbacterium insulae (EU239498)
99
Microbacterium thalassium (AB004713) 99
Pseudomonas brassicacearum
98
(AJ292381)
Variovorax soli (DQ432053)
97
Bacillus firmus (AJ717383)
99
Bacillus niacini (NR_024695)
96
Bacillus sp. (D84570)
Bacillus Pichinotyi (FJ639186)
98
Bosea eneae (DQ440825)
98
Cellulosimicrobium cellulans (AB098580)98
Lysobacter gummosus (FN313519)
99
Microbacterium flavescens (EU714363) 98
Nitratireductor basaltis (EU143347)
99
Pseudomonas fluorescens (AY538263) 98
Pseudomonas putida (AY918068)
98
Pseudomonas resinovorans (EU019983) 98
Pseudomonas sp. (AB453302)
99
Pseudomonas thivervalensis (EU834407) 99
Sporosarcina psychrophila (D16277)
98
Staphylococcus epidermidis (EU834240) 95
Variovorax soli (DQ432053)
98
Variovorax paradoxus (DQ256487)
97
Agrobacterium tumefaciens (FJ666055) 98
Lysobacter niabensis (DQ462461)
97
Microbacterium phyllosphaerae
98
(DQ328319)
Microbacterium insulae (EU239498)
97
Phenylobacterium koreense (AB166881) 99
Sinorhizobium meliloti (EU445252)
99
Variovorax paradoxus (EF641108)
99
Variovorax koreensis (EU827487)
98
Xanthomonas campestris (EU364838) 99
Fig. 1. The relative frequency of bacterial isolates belonging to different phylogenetic groups in the bulk soil, rhizosphere, and rhizoplane of tree peony plants.
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
FEMS Microbiol Lett 322 (2011) 15–24
23
Culturable bacterial community in the root of tree peony
than that of Fengdan, one reasonable explanation is that the
host genotypes influenced the distribution pattern of the
bacterial community in the root of tree peony plants. A
previous study reported that both bacterial and host genotypes influence endophytic colonization (Dong et al., 2003).
Further investigations will be necessary to verify whether
and how this distribution pattern is mediated by genetic
determinants of both partners.
Pseudomonas and Bacillus are considered important constituents in the root-associated microbial community, and
their ability to colonize the root surface, preventing the
development of plant pathogens and improving plant
growth, is well known (Rangarajan et al., 2001; Park et al.,
2005, 2008; Fett, 2006; Jorquera et al., 2011). We were
surprised that no members of these genera were found in
the rhizoplane of two tree peony varieties. In fact, no
members of Firmicutes were isolated in the rhizoplane. As
of now, it is unknown whether the absence of Firmicutes in
rhizoplane of tree peony plants is a common phenomenon.
But it is worth mentioning that tree peony is not only a kind
of ornamental plant but has also been used in traditional
Chinese medicine as an antimicrobial or anti-inflammatory,
whose main effective components are paeonol and paeoniflorin (Yan et al., 2004; Chung et al., 2007). At present, we
donot know whether and how the plant-associated bacterial
community is influenced by these antimicrobial components in tree peony plants.
This study provides basic information about the diversity
of bacteria associated with tree peony, a famous traditional
ornamental plant species in China. Despite some limitations
in this study of bacterial diversity, based on a culturedependent approach with eight isolation media, future work
is warranted to compare these results with those obtained
with culture-independent approaches.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (31070617), National Natural Science
Foundation of Shanghai (11ZR1436100), Program of
Shanghai Municipal Agricultural Commission (2008-10-4),
and Key Technologies R&D Program of Shanghai
(10391901200, 10dz2253700).
Authors’contribution
J.H. and Y.S. contributed equally to this work.
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