Arch Microbiol (2012) 194:1001–1012
DOI 10.1007/s00203-012-0836-8
ORIGINAL PAPER
Study on diversity of endophytic bacterial communities in seeds
of hybrid maize and their parental lines
Yang Liu • Shan Zuo • Liwen Xu •
Yuanyuan Zou • Wei Song
Received: 29 August 2011 / Revised: 23 May 2012 / Accepted: 30 July 2012 / Published online: 15 August 2012
Ó Springer-Verlag 2012
Abstract The seeds of plants are carriers of a variety of
beneficial bacteria and pathogens. Using the non-culture
methods of building 16S rDNA libraries, we investigated
the endophytic bacterial communities of seeds of four
hybrid maize offspring and their respective parents. The
results of this study show that the hybrid offspring Yuyu
23, Zhengdan958, Jingdan 28 and Jingyu 11 had 3, 33, 38
and 2 OTUs of bacteria, respectively. The parents Ye 478,
Chang 7-2, Zheng 58, Jing 24 and Jing 89 had 12, 36, 6, 12
and 2 OTUs, respectively. In the hybrid Yuyu 23, the
dominant bacterium Pantoea (73.38 %) was detected in its
female parent Ye 478, and the second dominant bacterium
of Sphingomonas (26.62 %) was detected in both its female
(Ye 478) and male (Chang 7-2) parent. In the hybrid
Zhengdan 958, the first dominant bacterium Stenotropho-
monas (41.67 %) was detected in both the female (Zheng
58) and male (Chang 7-2) parent. The second dominant
Communicated by Ursula Priefer.
Y. Liu  S. Zuo  Y. Zou  W. Song (&)
College of Life Sciences, Capital Normal University,
Beijing 100048, People’s Republic of China
e-mail: songwei@mail.cnu.edu.cn
Y. Liu
China National Research Institute of Food and Fermentation
Industries, Beijing 100027, People’s Republic of China
Y. Liu
China Center of Industrial Culture Collection,
China National Research Institute of Food and Fermentation
Industries, Beijing 100027, People’s Republic of China
L. Xu
Maize Research Center, Beijing Academy of Agriculture
and Forestry Sciences, Beijing 100097,
People’s Republic of China
bacterium Acinetobacter (9.26 %) was also the second
dominant bacterium of its male parent. In the hybrid
Jingdan 28, the second dominant bacterium Pseudomonas
(12.78 %) was also the second dominant bacterium of its
female parent, and its third dominant bacterium Sphingo-
monas (9.90 %) was the second dominant bacterium of its
male parent and detected in its female parent. In the hybrid
Jingyu 11, the first dominant bacterium Leclercia
(73.85 %) was the third dominant bacterium of its male
parent, and the second dominant bacterium Enterobacter
(26.15 %) was detected in its male parent. As far as we
know, this was the first research reported in China on the
diversity of the endophytic bacterial communities of the
seeds of various maize hybrids with different genotypes.
Keywords Hybrid maize  Seed endophytic bacteria 
Bacterial diversity  Culture-independent method
Abbreviation
CTAB Cetyltrimethylammonium bromide
Introduction
Endophytic bacteria are a class of endosymbiotic micro-
organisms that were able to colonize and healthfully
coexist with plant tissues (Kloepper and Beauchamp 1992).
Seeds act as the continuation organ of plants and serve as
an important means of agriculture production (Guan 2009),
but can also carry a variety of pathogens and beneficial
bacteria. Many studies have confirmed that the surface and
interior of seeds bear a variety of microbial organisms
(Nelson 2004). During seed germination, growth and
survival of these endophytic microbial communities and
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the microbial communities from the soil are facilitated
(Bacilio-Jiméne et al. 2001; Cottyn et al. 2001). The
microbes then may facilitate and interact with surrounding
plants, significantly impacting soil fertility and plant
growth (Barea et al. 2005).
Early studies have shown that the genotypes of plants
can impact the microorganisms coexisting with the plants.
Michiels et al. (1989) found that the genotype of a plant
controls the composition and the quantity of root exudates
and correlates with the quantity and activity of bacteria
colonizing the rhizosphere (e.g., azotobacter). Neal et al.
(1973) studied the species of microorganisms found in the
rhizosphere of different wheat genotypes and found that the
rhizosphere of mutant wheat contained different species
than the wild types. They suggested that the genotype of
the plants determined the species of microorganisms col-
onizing the rhizosphere.
Though there are many studies on rhizosphere microor-
ganism communities, up to now, few studies have focused
on microorganisms associated with seeds (Cankar et al.
2005) and even fewer have attempted to correlate endo-
phytic bacteria of maize seeds with their genotypes. In order
to understand the structure of endophytic bacterial com-
munities of different seed genotypes and explore the rela-
tionship of the endophytic bacterial community structures
of seeds of the filial generation of maize hybrids and their
parental lines, we constructed 16S rDNA libraries to iden-
tify the endophytic bacteria colonizing four combination
offspring of hybrid maize seeds and their respective parents.
Materials and methods
Maize seed sampling and surface sterilization
Seeds were collected from four hybrid combinations of
maize (Zea mays L.) (Yuyu 23, Zhengdan958, Jingdan 28,
Jingyu 11) and their parents (Ye 478, Chang 7-2, Zheng 58,
Fig. 1 Genetic relationships
among the four hybrid
combinations
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Arch Microbiol (2012) 194:1001–1012
Jing 24 and Jing 89) supplied by Professor Jiuran Zhao at
Beijing Academy of Agriculture and Forestry Sciences. The
genetic relationships among the samples are shown in Fig. 1.
All the samples were collected in March 2011 from the
Beijing Academy of Agriculture and Forestry Sciences
experimental plot in Sanya, Hainan (18.35774333340131 N,
109.18169975280762 E, southern China) and stored at 4 °C.
Maize seeds were washed with sterile water and
immersed in 70 % ethanol for 3 min. They were then
washed with fresh sodium hypochlorite solution (2.5 %
available Cl-) for 5 min, rinsed with 70 % alcohol for 30 s
and finally washed 5–7 more times with sterile water (Sun
et al. 2008). The aliquots of the final rinsing water were
spread on Luria–Bertani solid medium plates and cultured
for 3 days at 28 °C in order to confirm that the seeds were
sterilized and no seed surface bacteria remained. Only the
seed samples that were confirmed as sterile were used for
subsequent analysis.
DNA extraction and PCR amplification of the bacterial
16S rRNA gene
About 5.0 g of surface-sterilized maize seeds was frozen
with liquid nitrogen and quickly ground into a fine powder
with a precooled sterile mortar. Then, CTAB procedure
was used to extract the seed and bacterial DNA of all
samples (Sun et al. 2008). The DNA was then resuspended
in 30 lL sterile Milli-Q water.
The 16S rDNA of the indigenous bacteria within the
seeds was amplified using 799f (50 -AACAGGATTAGATA
CCCTG-30 ) and 1492r (50 -GGTTACCTTGTTACGACTT-30 )
as primers. These 2 primers were chosen because they can
separate bacterial and maize mitochondrial products (Sun
et al. 2008). The 50-lL PCR reaction mixture contained
50 ng of DNA extract, 1 9 Taq reaction buffer, 20 pmol of
each primer, 200 lmol of dNTP and 1.5 units Taq enzyme
(Ferments). Reaction procedure: initial denaturation at
94 °C for 5 min, denaturation at 94 °C for 1 min,
Arch Microbiol (2012) 194:1001–1012
annealing at 52 °C for 1 min, elongation at 72 °C for
1 min, after 30 circulations, extension at 72 °C for 10 min.
The temperature was then decreased to 52 °C to allow
annealing for 1 min then increased to 72 °C for 1 min of
elongation. After 30 cycles of the above, the temperature
was held at 72 °C for 10 min for extension. The PCR
products were then electrophoretically separated. The band
at approximately 750 bp was excised and purified using the
Wizard SV Gel and PCR Clean-up System (Promega) as
described by the manufacturer.
Construction of the16S rRNA gene clone library
The purified PCR products were ligated into the T3 vector
according to the protocol supplied by the manufacturer
(Transgen, China). Escherichia coli DH5a competent cells
(Transgen, China) were transformed with the ligation
products and spread onto LB agar plates with ampicillin
(100 lg/ml) and X-gal/IPTG on the surface for standard
blue and white screening. White colonies were randomly
picked and cultured in liquid LB over night.
Sequencing and phylogenetic analysis
Sequencing was performed on 100–150 randomly chosen
clones. Partial sequences of cloned 16S rRNA genes were
sequenced with an ABI 3730 DNA sequencer (ABI, USA).
All of the nucleotide sequences, approximately 700 bases,
were either compared with the NCBI database using
BLASTN or aligned by the identification analysis of
EzTaxon server 2.1 (Chun et al. 2007). Sequences with
[97 % similarity were assigned to the same species.
Results
Electrophoresis of the plant seed and the endophytic bacteria
ligation products showed two bands. One lied between 1,000
and 1,500 bp, which corresponds with maize mitochondrial
18S rDNA; the other was found between 700 and 800 bp,
corresponding with endophytic bacteria 16S rDNA. We
amplified the target fragments of 100–150 clones from each
library. The sequence information was submitted to the
GenBank accession (accession No. JN167639–JN167786,
excluding JN167649, JN167668, JN167692 and JN167673).
The combination offspring of hybrid maize, Yuyu 23,
Zhengdan958, Jingdan 28 and Jingyu 11 had 3, 33, 38 and
2 OTUs, respectively. The parents Ye 478, Chang 7-2,
Zheng 58, Jing 24 and Jing 89 had 12, 36, 6, 12 and 2
OTUs, respectively (Tables 1, 2, 3, 4,5, 6, 7, 8, 9).
In the Yuyu 23, the hybrid offspring of Ye 478 9 Chang
7-2, the endophytic bacterial community was less species
rich than and the dominant bacteria were inconsistent with
1003
those of the two parents. Yuyu 23 seeds had only 2 bacterial
OTUs, and the dominant bacteria genera were Pantoea
(73.38 %) and Sphingomonas (26.62 %). The male and
female parent seeds, containing 12 and 36 OTUs, respec-
tively were richer in endophytic bacterial species than their
offspring. For the female parent, Ye 478, the dominant
bacteria genera were Leclercia (50.00 %), Tatumella
(21.09 %) and Enterobacter (13.28 %), while those of the
male parent, Chang 7-2, were Roseateles (36.67 %), Aci-
netobacter (15.56 %) and Burkholderia (10.00 %). Though
the first (Pantoea) and second (Sphingomonas) dominant
bacterial genera of the offspring was not dominant in either
of the parents, Pantoea was detected in the female parent,
and Sphingomonas was detected in both the female and
male parent (Tables 1, 2, 6, 10).
In Zhengdan 958, the hybrid combination of Zheng
58 9 Chang 7-2, the seeds had more species of endophytic
bacteria than and some similarities in dominant bacteria
with the parents. Zhengdan 958 seeds contained 33 OTUs,
while the female and male parent seeds contained 7 and 36
OTUs, respectively. The dominant bacteria of Zhengdan
958 seeds were Sphingomonas (41.67 %), Bacillus/Acine-
tobacter (9.26 %) and Leclercia (7.41 %). The dominant
bacteria genera in the female parent, Zheng 58, were
Klebsiella (93.23 %), Pseudomonas (3.01 %) and Steno-
trophomonas (2.26 %), while those of male parent, Chang
7-2, were Roseateles (36.67 %), Acinetobacter (15.56 %)
and Burkholderia (10.00 %). In this combination, the sec-
ond dominant genus (Acinetobacter) of the hybrid off-
spring was consistent with the second dominant bacteria of
its male parent, and the first dominant bacterium (Steno-
trophomonas) of the offspring was detected in both the
female and male parent (Tables 2, 3, 7, 10).
The hybridization of Zheng 58 9 Jing 24 resulted in the
offspring, Jingdan 28. Jingdan 28 had more species of
endophytic bacteria than its parents and similar dominant
genera to its parents. The offspring seeds of Jingdan 28
contained 38 OTUs, compared to 7 and 12 OTUs for the
female and male parent, respectively. For Jingdan 28, the
dominant bacteria genera were Roseateles (31.68 %),
Pseudomonas (12.87 %) and Sphingomonas (9.90 %). As
for the female parent, Zheng 58, the dominant bacteria
genera were Klebsiella (93.23 %), Pseudomonas (3.01 %)
and Stenotrophomonas (2.26 %), while those of the male
parent, Jing 24, were Serratia (68.50 %), Sphingomonas
(18.11 %) and Luteibacter/Leclercia (3.15 %). In this
combination, the second dominant bacterium (Pseudomo-
nas) of Jingdan 28 was consistent with the second domi-
nant bacterium of its female parent, and the hybrid
offspring’s third dominant bacterium (Sphingomonas) was
consistent with the second dominant bacterium of its male
parent. Furthermore, all of Jingdan 28’s dominant bacteria
were detected in its female parent (Tables 3, 4, 8, 10).
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Table 1 Distribution of 16S rRNA clones detected from endophytes of Ye 478

Group No. of Genus Strain No. of Percentage of Closest NCBI match Percentage GenBank
OTUs clones total clones of indentity accession no.

Proteobacteria 10 Tatumella YA148 27 21.09 Tatumella morbirosei (EU344769) 99 JN167639

Leclercia YA114 64 50.00 Leclercia adecarboxylata 99 JN167641
(AB273740)
Enterobacter YA25 1 0.78 Enterobacter dissolvens (Z96079) 98 JN167642
YA103 16 12.50 Enterobacter cancerogenus 99 JN167646
(Z96078)
Serratia YA38 2 1.56 S. marcescens (AB061685) 99 JN167643
Erwinia YA15 3 2.34 Erwinia cypripedii (U80201) 99 JN167644
YA71 1 0.78 Erwinia aphidicola (AB273744) 99 JN167651
Sphingomonas YA22 2 1.56 Sphingomonas yanoikuyae 99 JN167645
(EU009209)
Pantoea YA126 1 0.78 Pantoea anthophila (EF688010) 99 JN167647
YA78 9 7.03 P. dispersa (DQ504305) 99 JN167640
Firmicutes 2 Oxalophagus YA133 1 0.78 Oxalophagus oxalicus (Y14581) 99 JN167648
Paenibacillus YA3 1 0.78 Paenibacillus nanensis (AB265206) 94 JN167650

Jingyu 11 was the hybridization of Jing 89 9 Jing 24.
The offspring seed of Jingyu 11 had fewer species of
endophytic bacteria, but similar dominant bacterium gen-
era relative to its parents. Jing 11 seeds contained only 2
OTUs. The female and male parent seeds’ endophytic
bacteria were made up of 2 and 12 OTUs, respectively. For
Jingyu 11, the dominant bacteria genera were Leclercia
(73.85 %) and Enterobacter (26.15 %). In the female
parent, Jing 89, the dominant bacteria genera were Pantoea
(99.16 %) and Sphingomonas (0.84 %). In the male parent,
Jing 24, the dominant bacteria genera were Serratia
(68.50 %), Sphingomonas (18.11 %) and Luteibacter/
Leclercia (3.15 %). In this combination, the first dominant
bacterium (Leclercia) of the hybrid offspring was con-
sistent with the third dominant bacterium of the male
parent, and the second dominant bacterium (Enterobacter)
of the offspring was detected in the male parent (Tables 4,
5, 9, 10).
The hybrid offspring Yuyu 23 and Zhengdan 958 had
the same male parent (Chang 7-2), but different female
parents. The seeds of Zhengdan 958 were notably rich in
endophytic bacteria species. Sphingomonas was the domi-
nant endophytic bacterial genus for both offspring, and it
was detected in the male parent. The genus, Pantoea was
also detected in both Yuyu 23 and Zhengdan 958
(Tables 2, 6, 7).
The hybrid offspring of Zhengdan 958 and Jingdan 28
had the same female parent (Zheng 58), but with different
male parents. Both offspring had more endophytic bacteria
species than their parents. Sphingomonas, which was also
present in the female parent, was the dominant endophytic
bacterial genus for both Zhengdan 958 and Jingdan 28.
The bacterial genera Shigella, Burkholderia, Acidovorax,
Acinetobacter, and Serratia were present in both offspring
(Tables 3, 7, 8).
The hybrid offspring Jingdan 28 and Jingyu 11 both had
Jing 24 as their male parent. There were clear differences
between the endophytic bacteria of these 2 hybrids. There
were no similarities in the dominant endophytic genera of
the 2 hybrids, but the dominant endophytic bacterium
(Sphingomonas) of Jingdan28 and the dominant endophytic
bacterium of Jingyu 11 were both detected in the male
parent (Tables 4, 8, 9, 10).
There were obvious differences among the four hybrids
and their male parent seeds in number and species of
endophytic bacteria, but most dominant endophytic bacte-
ria of the offspring were also detected in their parental
seeds. When comparing the endophytic bacteria, especially
the dominant endophytic species found in hybrids with
genetic correlations have some relevance with their parents
(Tables 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).
Discussion
Seeds are carriers of both microphyte parental genes (Guan
2009) and a variety of beneficial bacteria and pathogens.
These microorganisms originate from various microbial
communities born on the seed surface (Nelson 2004). To
the best of our knowledge, studies on the endophytic bac-
teria of maize are much more less than rice. Using culture
methods, Rijavec et al. (2007) identified the endophytic
bacteria genera released during germination of four types
of maize seeds. Johnston-Monje and Raizada (2011) found
that seed endophyte community composition varied in
relation to plant host phylogeny, there was a core

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Table 2 Distribution of 16S rRNA clones detected from endophytes of Chang 7-2
Group No. of Genus Strain No. of Percentage of Closest NCBI match Percentage GenBank
OTUs clones total clones of indentity accession no.

Proteobacteria 25 Bosea CHB143 1 1.11 Bosea vestrisii (AF288306) 99 JN167652

Rheinheimera CHB141 1 1.11 Rheinheimera soli 99 JN167653
(EF575565)
Acinetobacter CHB138 3 3.33 Acinetobacter baumannii 99 JN167654
(ACQB01000091)
CHB121 9 10.00 Acinetobacter johnsonii 99 JN167660
(X81663)
CHB32 1 1.11 Acinetobacter beijerinckii 99 JN167680
(AJ626712)
CHB22 1 1.11 Acinetobacter schindleri 99 JN167685
(AJ278311)
Roseateles CHB85 31 34.44 Roseateles depolymerans 99 JN167655
(AB003626)
CHB132 2 2.22 Roseateles terrae 98 JN167663
(AM501445)
Burkholderia CHB13 9 10.00 Burkholderia diffusa 99 JN167657
(AM747629)
Sphingopyxis CHB106 1 1.11 Sphingopyxis panaciterrae 99 JN167658
(AB245353)
Massilia CHB24 1 1.11 Massilia aerolata 99 JN167682
(EF688526)
CHB129 1 1.11 Massilia albidiflava 98 JN167661
(AY965999)
Pseudomonas CHB128 1 1.11 Pseudomonas poae 99 JN167662
(AJ492829)
Ancylobacter CHB131 1 1.11 Ancylobacter rudongensis 94 JN167664
(AY056830)
Stenotrophomonas CHB135 1 1.11 Stenotrophomonas pavanii 100 JN167665
(FJ748683)
Shigella CHB117 4 4.44 Shigella flexneri (X96963) 99 JN167666
Bdellovibrio CHB71 1 1.11 Bdellovibrio bacteriovorus 91 JN167671
(BX842601)
Enterobacter CHB58 1 1.11 E. cancerogenus (Z96078) 99 JN167674
Enhydrobacter CHB44 1 1.11 Enhydrobacter aerosaccus 99 JN167675
(AJ550856)
Variovorax CHB63 1 1.11 Variovorax boronicumulans 99 JN167676
(AB300597)
Oceanibaculum CHB46 1 1.11 Oceanibaculum pacificum 92 JN167677
(FJ463255)
Sphingomonas CHB25 1 1.11 S. yanoikuyae (EU009209) 100 JN167683
Devosia CHB21 1 1.11 Devosia riboflavina 97 JN167684
(AJ549086)
Escherichia CHB14 1 1.11 E. coli (CP001164) 99 JN167686
Sphingosinicella CHB8 1 1.11 Sphingosinicella 94 JN167688
xenopeptidilytica
(AY950663)
Firmicutes 3 Paenibacillus CHB40 1 1.11 Paenibacillus daejeonensis 99 JN167679
(AF391124)
CHB15 1 1.11 Paenibacillus xylanilyticus 99 JN167687
(AY427832)
Sediminibacillus CHB5 1 1.11 Sediminibacillus halophilus 98 JN167689
(AM905297)

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Table 2 continued
Group No. of Genus Strain No. of Percentage of Closest NCBI match Percentage GenBank
OTUs clones total clones of indentity accession no.

Actinobacteria 3 Corynebacterium CHB77 2 2.22 Corynebacterium 98 JN167659

pseudogenitalium
(ABYQ01000237)
Nocardia CHB72 1 1.11 Nocardia soli (AF430051) 99 JN167670
Lentzea CHB50 1 1.11 Lentzea flaviverrucosa 100 JN167672
(AF183957)
Bacteroidetes 3 Flavobacterium CHB145 1 1.11 Flavobacterium degerlachei 98 JN167656
(AJ557886)
CHB82 1 1.11 Flavobacterium aquatile 97 JN167669
(M62797)
Chryseobacterium CHB47 1 1.11 Chryseobacterium hominis 99 JN167678
(AM261868)
Uncultured 2 – CHB119 2 2.22 Uncultured bacterium 96 JN167667
bacteria (EU335174)
– CHB29 1 1.11 Uncultured bacterium 95 JN167681
(EU276548)

Table 3 Distribution of 16S rRNA clones detected from endophytes of Zheng 58

Group No. of Genus Strain No. of Percentage of Closest NCBI match Percentage of GenBank
OTUs clones total clones indentity accession no.

Proteobacteria 6 Klebsiella ZC150 87 65.41 Klebsiella variicola 100 JN167690

(AJ783916)
ZC138 37 27.82 K. pneumoniae 99 JN167691
(ACZD01000038)
Pseudomonas ZC73 4 3.01 Pseudomonas 98 JN167693
plecoglossicida
(AB009457)
Stenotrophomonas ZC37 3 2.26 S. pavanii (FJ748683) 100 JN167694
Sphingomonas ZC42 1 0.75 Sphingomonas 99 JN167695
echinoides (AJ012461)
Rhizobium ZC25 1 0.75 Rhizobium massiliae 100 JN167696
(AF531767)

microbiota of endophytes that was conserved in maize
seeds across boundaries of evolution, ethnography and
ecology. This study is an attempt to use non-culture
methods to study the diversity of endophytic bacterial
communities associated with the seeds of the new type
maize hybrids and their parents which autonomously cul-
tured by China. The purpose of the research is to investi-
gate the influence of maize seed genotype on the structure
of seed endophytic bacterial communities.
The results show clear differences in number and spe-
cies of endophytic bacteria among the seeds of the four
offspring hybrids and their parents. One of the reasons for
these differences may be variability in genotype among the
plants investigated. Simon et al. (2001) found the growth of
inherent and inoculated bacteria differed on the seeds of
different tomato genotypes. Adams and Kloepper (2002)
investigated the impact of cotton plant genotype on the
inherent bacterial population of their seeds, seedling stems
and root tissues. They found that cotton plants have
the capacity to carry endophytic bacterial communities
immediately after seed germination and that in the process
of seed germination and seedling development, the distinct
genetic, morphological and physiological characteristics of
individual cotton cultivars led to differences in the endo-
phytic bacterial community structure among the cultivars.
Picard and Bosco (2006) found that the filial generation of
hybrid maize contains more protein than their parents,
which results in attraction of more pseudomonas to the
filial generation roots. Xiao et al. (1995) reported that
dominance complementation is the major genetic basis of

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Table 4 Distribution of 16S rRNA clones detected from endophytes of Jing 24

Group No. of Genus Strain No. of Percentage of Closest NCBI match Percentage of GenBank
OTUs clones total clones indentity accession no.

Proteobacteria 11 Serratia J24D34 87 68.50 S. marcescens 99 JN167697

(AB061685)
Sphingomonas J24D36 21 16.54 S. echinoides 99 JN167698
(AJ012461)
J24D56 2 1.57 Sphingomonas 99 JN167701
dokdonensis
(DQ178975)
Pantoea J24D42 1 0.79 P. dispersa (DQ504305) 99 JN167699
Luteibacter J24D54 4 3.15 Luteibacter anthropi 100 JN167700
(FM212561)
Burkholderia J24D61 1 0.79 Burkholderia gladioli 100 JN167702
(EU024168)
Leclercia J24D13 4 3.15 L. adecarboxylata 99 JN167703
(AB273740)
Tepidimonas J24D72 1 0.79 Tepidimonas aquatic 99 JN167705
(AY324139)
Tatumella J24D131 1 0.79 T. morbirosei 99 JN167706
(EU344769)
Enterobacter J24D143 1 0.79 E. cancerogenus 99 JN167707
(Z96078)
Thermomonas J24D120 1 0.79 Thermomonas brevis 97 JN167708
(AJ519989)
Firmicutes 1 Lactobacillus J24D23 3 2.36 Lactobacillus iners 99 JN167704
(ACLN01000018)

Table 5 Distribution of 16S rRNA clones detected from endophytes of Jing 89

Group No. of Genus Strain No. of Percentage of Closest NCBI Percentage of GenBank
OTUs clones total clones match indentity accession no.

Proteobacteria 2 Pantoea J89E143 118 99.16 P. dispersa 100 JN167709

(DQ504305)
Sphingomonas J89E132 1 0.84 S. echinoides 99 JN167710
(AJ012461)

Table 6 Distribution of 16S rRNA clones detected from endophytes of Yuyu 23

Group No. of Genus Strain No. of Percentage of Closest NCBI Percentage of GenBank
OTUs clones total clones match indentity accession no.

Proteobacteria 3 Pantoea YYIb34 34 24.46 P. dispersa 100 JN167784

(DQ504305)
YYI105 68 48.92 P. agglomerans 99 JN167785
(AJ233423)
Sphingomonas YYI146 37 26.62 S. echinoides 99 JN167786
(AJ012461)

hybridization and many hybrid offspring possess traits
superior to their parents. In the present study, all offspring
of the four maize hybrids had strong agronomic traits
(Table 11) (Sun et al. 2005; San et al. 2007; Chen et al.
2009). Hence, we can infer that the differences in
nutritional structure among the genotypes of maize seeds
are probably one of the key reasons for differences in
endophytic bacterial communities.
It should be noted that the factors that affect the endo-
phytic bacteria of plant seeds, especially the dominant

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Table 7 Distribution of 16S rRNA clones detected from endophytes of Zhengdan 958
Group No. of Genus Strain No. of Percentage Closest NCBI match Percentage GenBank
OTUs clones of total of indentity accession no.
clones

Proteobacteria 23 Sphingomonas ZDF3 45 41.67 S. echinoides (AJ012461) 99 JN167713

Shigella ZDF7 3 2.78 S. flexneri (X96963) 99 JN167714
Leclercia ZDF12 8 7.41 L. adecarboxylata (AB273740) 99 JN167716
Pseudoxanthomonas ZDF127 1 0.93 Pseudoxanthomonas kaohsiungensis (AY650027) 99 JN167717
Psychrobacter ZDF134 1 0.93 Psychrobacter pulmonis (AJ437696) 99 JN167718
Variovorax ZDF132 1 0.93 V. boronicumulans (AB300597) 99 JN167720
Enterobacter ZDF136 2 1.85 Enterobacter sp. (GU814270) 99 JN167721
Microvirga ZDF118 1 0.93 Microvirga aerophilus (GQ421848) 97 JN167727
ZDF103 1 0.93 Microvirga aerilata (GQ421849) 97 JN167734
Erwinia ZDF117 2 1.85 E. aphidicola (AB273744) 99 JN167725
Methylobacterium ZDF78 1 0.93 Methylobacterium platani (EF426729) 98 JN167729
Tatumella ZDF83 2 1.85 T. morbirosei (EU344769) 99 JN167730
Burkholderia ZDF91 1 0.93 Burkholderia phytofirmans (CP001053) 99 JN167732
ZDF109 3 2.78 Burkholderia sp. (AM747629) 100 JN167723
Acidovorax ZDF98 1 0.93 Acidovorax temperans (AF078766) 99 JN167733
Serratia ZDF38 1 0.93 S. marcescens (AJ233431) 100 JN167743
ZDF41 3 2.78 Serratia ureilytica (AJ854062) 98 JN167737
Acinetobacter ZDF64 1 0.93 A. beijerinckii (AJ626712) 99 JN167738
ZDF13 1 0.93 Acinetobacter junii (AM410704) 98 JN167739
ZDF114 6 5.56 A. johnsonii (X81663) 100 JN167724
ZDF119 2 1.85 Acinetobacter kyonggiensis (FJ527818) 98 JN167726
Halomonas ZDF37 1 0.93 Halomonas daqingensis (EF121854) 99 JN167741
Pantoea ZDF49 1 0.93 P. dispersa (DQ504305) 100 JN167736
Firmicutes 7 Lactobacillus ZDF4 1 0.93 L. iners (ACLN01000018) 99 JN167712
Bacillus ZDF9 10 9.26 Bacillus aryabhattai (EF114313) 99 JN167715
Staphylococcus ZDF138 1 0.93 Staphylococcus hominis (X66101) 99 JN167722
ZDF107 1 0.93 Staphylococcus capitis (L37599) 99 JN167728
Finegoldia ZDF106 1 0.93 Finegoldia magna (AF542227) 99 JN167735
Ruminococcus ZDF21 1 0.93 Ruminococcus bromii (L76600) 92 JN167740
Aerococcus ZDF39 1 0.93 Aerococcus urinaeequi (D87677) 99 JN167742
Actinobacteria 2 Propioniciclava ZDF1 1 0.93 Propioniciclava tarda (AB298731) 95 JN167711
Propionibacterium ZDF133 1 0.93 Propionibacterium acnes (AE017283) 99 JN167719
Uncultured bacteria 1 – ZDF86 1 0.93 Uncultured bacterium (EU335174) 94 JN167731

bacterial genera, depend not only on the genotype of plants,
but also on the seed itself. Other important variables
influencing the bacterial communities of seeds and their
succession may be seed shape, different parts of tissue and
development and germination stage (Zou et al. 2011).
The above results also show that the dominant endo-
phytic bacteria of the seeds of the four hybrid maize off-
spring for this study were also detected in parental seeds.
Additionally, the endophytic bacterial communities in the
seeds of genetically related offspring had similar species
and, in particular, dominant genera compositions. We have
illustrated two possible reasons for these trends: (1) The
combined hybrid maize seeds are genetically related; (2) in
this study, all seed samples were collected at the same period
in the same experimental field, so the environmental factors
that may influence bacterial communities were relatively
uniform. The present study selected a combination of
genetically related hybrid maize. It was found that most
dominant endophytic of hybrid offspring were detected in
the parental seeds, indicating that there is a continuity of
endophytic bacteria from the parental to the offspring seed.
Reports have shown that endophytic bacteria colonize plant
seeds and become an important source of endophytic bac-
teria for the growing plants (Feng and Song 2001). Recently,
we found that the bacteria in the hybrid seeds with the
contents more than 5 % could also be detected in their male
or female parental seeds. Endophytic bacterial communities
of offspring seeds are likely affected by the parental strains
(results unpublished). The structures of endophytic bacterial
communities of genetically related offspring, namely hybrid
offspring with the same father or the same mother, have
some similarities due to their common parent strains.
Soil environment in which a plant grows is an important
factor for bacterial communities associated with the plant

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Table 8 Distribution of 16S rRNA clones detected from endophytes of Jingdan 28

Group No. of Genus Strain No. of Percentage of Closest NCBI match Percentage GenBank
OTUs clones total clones of indentity accession no.

Proteobacteria 30 Brevundimonas JDGb36 1 0.99 Brevundimonas diminuta 99 JN167744

(GL883089)
JDG95 1 0.99 Brevundimonas naejangsanensis 99 JN167764
(FJ544245)
Sphingomonas JDGb33 8 7.92 S. echinoides (AJ012461) 99 JN167745
JDG114 1 0.99 Sphingomonas koreensis (AF131296) 98 JN167756
JDG117 1 0.99 Sphingomonas humi (AB220146) 96 JN167758
Acidovorax JDGb6 1 0.99 Acidovorax facilis (AF078765) 99 JN167746
JDG113 1 0.99 A. temperans (AF078766) 97 JN167757
Shinella JDG133 1 0.99 Shinella zoogloeoides (X74915) 99 JN167747
Roseateles JDG146 29 28.71 R. depolymerans (AB003626) 99 JN167748
JDG141 3 2.97 R. terrae (AM501445) 99 JN167752
Rheinheimera JDG145 1 0.99 Rheinheimera chironomi 99 JN167749
(DQ298025)
JDG5 1 0.99 R. soli (EF575565) 99 JN167775
Acinetobacter JDG143 7 6.93 A. johnsonii (X81663) 99 JN167750
JDG73 2 1.98 A. schindleri (AJ278311) 99 JN167761
JDG97 1 0.99 Acinetobacter lwoffii (X81665) 99 JN167765
Pseudomonas JDG142 13 12.87 Pseudomonas stutzeri (U26262) 99 JN167751
Thermomonas JDG34 2 1.98 Thermomonas koreensis (DQ154906) 99 JN167753
Burkholderia JDG42 4 3.96 Burkholderia sp. (AM747629) 99 JN167754
Shigella JDG100 4 3.96 S. flexneri (X96963) 99 JN167760
Cellvibrio JDG1 1 0.99 Cellvibrio mixtus (AF448515) 99 JN167766
Serratia JDG99 1 0.99 S. marcescens (AJ233431) 100 JN167767
Thiobacillus JDG43 1 0.99 Thiobacillus aquaesulis (U58019) 93 JN167768
Luteimonas JDG44 1 0.99 Luteimonas aestuarii (EF660758) 98 JN167769
Sphingosinicella JDG67 1 0.99 Sphingosinicella sp. (FJ442859) 96 JN167772
Acidithiobacillus JDG68 1 0.99 Acidithiobacillus albertensis (AJ459804) 100 JN167773
Curvibacter JDG2 1 0.99 Curvibacter gracilis (AB109889) 100 JN167774
Devosia JDG19 1 0.99 Devosia insulae (EF012357) 97 JN167777
Cupriavidus JDG24 1 0.99 Cupriavidus gilardii (AF076645) 98 JN167778
Methylobacterium JDG31 1 0.99 Methylobacterium rhodesianum 99 JN167779
(AB175642)
Dokdonella JDG30 1 0.99 Dokdonella sp. (EU685334) 98 JN167780
Firmicutes 1 Desemzia JDG94 1 0.99 Desemzia incerta (Y14650) 99 JN167763
Actinobacteria 3 Kocuria JDG55 1 0.99 Kocuria rosea (X87756) 99 JN167770
Nocardia JDG62 1 0.99 Nocardia ignorata (AJ303008) 99 JN167771
Pseudonocardia JDG10 1 0.99 Pseudonocardia aurantiaca (FR749916) 99 JN167776
Bacteroidetes 4 Flavobacterium JDG102 1 0.99 Flavobacterium johnsoniae (CP000685) 99 JN167755
JDG88 1 0.99 Flavobacterium mizutaii (AJ438175) 99 JN167762
Flavisolibacter JDG28 1 0.99 Flavisolibacter ginsengiterrae 98 JN167781
(AB267476)
Sphingobacterium JDG129 1 0.99 Sphingobacterium daejeonense 99 JN167759
(AB249372)

(Jefferey et al. 1999). Correspondingly, the main species of
bacteria detected in this study were common soil bacteria.
There are two factors that bacteria in the soil environment
become the source of endophytic bacteria of plants. On the
one hand, the roots of plants are exposed to bacteria in the
soil environment during growth and development, which
provides opportunities for bacteria to enter into the plants.
On the other hand, certain genes of plants and bacteria
determine the interaction between the two and determine
whether the bacteria are colonized in the plant or not. The
unique characteristics of different types of plants will allow
different species of bacteria to colonize their body or body
surface (Hardoim et al. 2008). Physiological characteristics
of the plant also affect the structure of endophytic bacterial

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Table 9 Distribution of 16S rRNA clones detected from endophytes of Jingyu 11

Group No. of Genus Strain No. of Percentage of Closest NCBI match Percentage of GenBank
OTUs clones total clones indentity accession no.

Proteobacteria 2 Leclercia JYH3 96 73.85 L. adecarboxylata 99 JN167782

(AB273740)
Enterobacter JYH4 34 26.15 E. cancerogenus 99 JN167783
(Z96078)

Table 10 Comparison of dominant genera from nine seed samples

Ye 478 Chang 7-2 Zheng 58 Jing 24 Jing 89 Zhengdan958 Jingdan 28 Jingyu 11 Yuyu 23

The first Leclercia Roseateles Klebsiella Serratia Pantoea Sphingomonas Roseateles Leclercia Pantoea
dominant (50.00 %) (36.67 %) (93.23 %) (68.50 %) (99.16 %) (41.67 %) (31.68 %) (73.85 %) (73.38 %)
genera
The second Tatumella Acinetobacter Pseudomonas Sphingomonas Sphingomonas Bacillus/ Pseudomonas Enterobacter Sphingomonas
dominant (21.09 %) (15.56 %) (3.01 %) (18.11 %) (0.84 %) Acinetobacter (12.87 %) (26.15 %) (26.62 %)
genera (9.26 %)
The third Enterobacter Burkholderia Stenotrophomonas Luteibacter/ – Leclercia Sphingomonas – –
dominant (13.28 %) (10.00 %) (2.26 %) Leclercia (7.41 %) (9.90 %)
genera (3.15 %)

Table 11 Agronomic traits of four maize hybrids

Seed Agronomic traits
Crude Crude Crude fat Lysine
protein (%) starch (%) (%) (%)

Yuyu 23 10.00 73.12 4.55 0.28 Widely adaptable, high yielding, high quality, pathogen resistant and highly
fecund
Zhengdan 9.33 73.02 3.98 0.25 High and stable yields, good quality, pathogen resistance, good fecundity and
958 good drought and heat tolerance
Jingdan 9.47 74.82 4.01 0.25 Good disease and lodging resistance and green-keeping capability
28
Jingyu 11 8.17 75.32 4.17 0.26 High yielding, high lodging resistance, disease resistance and shade and high
moisture tolerance

communities (van Overbeek and van Elsas 2008). The
present study found that the dominant bacteria genera (i.e.,
Sphingomonas, Leclercia, Pantoea, Pseudomonas, Acine-
tobacter, Roseateles and Enterobacter) of maize seeds
grown in a relatively uniform environment tend to be found
in relative abundance in that environment, these species
could be more adaptable to the unique physiological
environment within the mature maize seeds of the hybrid
type they were found in. This physiological environment is
determined by the species and genotype of the seed.
Among the detected dominant bacteria genera (Pseu-
domonas, Acinetobacter, Bacillus, Sphingomonas, Entero-
bacter, Burkholderia and Klebsiella), some can be plant
growth-promoting bacteria (Sturz 1995; Videira et al.
2009; Lucy et al. 2004; Liu et al. 2011), which may directly
or indirectly affect the growth and development of plants
(Feng and Song 2001). The dominant bacterium Pantoea
dispersa identified in this study produces an esterase that
irreversibly degrades pathogenic substance and thus
restricts the growth of plant pathogens (Zhang and Birch
1997). Pantoea agglomerans are able to fix nitrogen and
secreted hormone of the plant (Feng et al. 2003). Serratia
marcescens are able to produce antibiotics providing pro-
tection against some plant pathogens (Wei et al. 1996).
Klebsiella pneumoniae NG14, which has been isolated
from rice root, appears to promote plant growth through
synthesis of auxin IAA from indole-3-pyruvate, biological
nitrogen fixation and colonization of the root surface and
root vascular tissue within the cavity of the rice seedlings
(Liu et al. 2011). These plant growth-promoting bacteria
can be used to increase yield, improve quality, enhance
pathogen resistance and shortened growth cycles through
biological nitrogen fixation, secretion of plant growth
regulators and production of antibiotics. At the same time,

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the beneficial effects of these growth-promoting endo-
phytic bacteria on plants likely improve the environmental
conditions within the plant for the same bacteria causing a
positive feedback loop.
This study is the first attempt to employ culture-inde-
pendent techniques to investigate the endophytic bacteria of
the seeds of hybrid maize offspring and their parents. Our
results provide a solid foundation for further study of the
correlation of endophytic bacteria community with structure
seed genotypes. This provides the necessary baseline infor-
mation for further studies of the relationships and interac-
tions between endophytic bacteria and their host seeds and
may provide a new starting point for investigations of plant
heterosis. Further research on correlations among the
endophytic bacteria communities of different seed geno-
types and seeds of hybrid offspring and their parents and the
agronomic traits of these bacteria is necessary.
Acknowledgments This work was supported by the National Natural
Science Foundation of China (No. 30770069), the Science Foundation of
Beijing (No. 5092004). National Science and Technology Support Plans
of China (2012BAK17B11) and International Science and Technology
Cooperation Projects of Beijing (Z111105054611011). We would like to
thank Professor Jiuran Zhao and Fengge Wang at Beijing Academy of
Agriculture and Forestry Sciences for their assistance with supplying the
seed sample of maize. We would also like to thank Christine Verhille
at the University of British Columbia for her assistance with English
language and grammatical editing of the manuscript.
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