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Archives of Microbiology ISSN 0302-8933 Arch MicrobiolDOI 10.1007/s00203-012-0836-8

Study on diversity of endophytic bacterialcommunities in seeds of hybrid maize andtheir parental lines

Yang Liu, Shan Zuo, Liwen Xu,Yuanyuan Zou & Wei Song

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ORIGINAL PAPER

Study on diversity of endophytic bacterial communities in seedsof 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

� 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

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

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

123

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DOI 10.1007/s00203-012-0836-8

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the microbial communities from the soil are facilitated

(Bacilio-Jimene 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,

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,

Fig. 1 Genetic relationships

among the four hybrid

combinations

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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

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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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

Table 1 Distribution of 16S rRNA clones detected from endophytes of Ye 478

Group No. of

OTUs

Genus Strain No. of

clones

Percentage of

total clones

Closest NCBI match Percentage

of indentity

GenBank

accession no.

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

Leclercia YA114 64 50.00 Leclercia adecarboxylata(AB273740)

99 JN167641

Enterobacter YA25 1 0.78 Enterobacter dissolvens (Z96079) 98 JN167642

YA103 16 12.50 Enterobacter cancerogenus(Z96078)

99 JN167646

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(EU009209)

99 JN167645

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

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Table 2 Distribution of 16S rRNA clones detected from endophytes of Chang 7-2

Group No. of

OTUs

Genus Strain No. of

clones

Percentage of

total clones

Closest NCBI match Percentage

of indentity

GenBank

accession no.

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

Rheinheimera CHB141 1 1.11 Rheinheimera soli(EF575565)

99 JN167653

Acinetobacter CHB138 3 3.33 Acinetobacter baumannii(ACQB01000091)

99 JN167654

CHB121 9 10.00 Acinetobacter johnsonii(X81663)

99 JN167660

CHB32 1 1.11 Acinetobacter beijerinckii(AJ626712)

99 JN167680

CHB22 1 1.11 Acinetobacter schindleri(AJ278311)

99 JN167685

Roseateles CHB85 31 34.44 Roseateles depolymerans(AB003626)

99 JN167655

CHB132 2 2.22 Roseateles terrae(AM501445)

98 JN167663

Burkholderia CHB13 9 10.00 Burkholderia diffusa(AM747629)

99 JN167657

Sphingopyxis CHB106 1 1.11 Sphingopyxis panaciterrae(AB245353)

99 JN167658

Massilia CHB24 1 1.11 Massilia aerolata(EF688526)

99 JN167682

CHB129 1 1.11 Massilia albidiflava(AY965999)

98 JN167661

Pseudomonas CHB128 1 1.11 Pseudomonas poae(AJ492829)

99 JN167662

Ancylobacter CHB131 1 1.11 Ancylobacter rudongensis(AY056830)

94 JN167664

Stenotrophomonas CHB135 1 1.11 Stenotrophomonas pavanii(FJ748683)

100 JN167665

Shigella CHB117 4 4.44 Shigella flexneri (X96963) 99 JN167666

Bdellovibrio CHB71 1 1.11 Bdellovibrio bacteriovorus(BX842601)

91 JN167671

Enterobacter CHB58 1 1.11 E. cancerogenus (Z96078) 99 JN167674

Enhydrobacter CHB44 1 1.11 Enhydrobacter aerosaccus(AJ550856)

99 JN167675

Variovorax CHB63 1 1.11 Variovorax boronicumulans(AB300597)

99 JN167676

Oceanibaculum CHB46 1 1.11 Oceanibaculum pacificum(FJ463255)

92 JN167677

Sphingomonas CHB25 1 1.11 S. yanoikuyae (EU009209) 100 JN167683

Devosia CHB21 1 1.11 Devosia riboflavina(AJ549086)

97 JN167684

Escherichia CHB14 1 1.11 E. coli (CP001164) 99 JN167686

Sphingosinicella CHB8 1 1.11 Sphingosinicellaxenopeptidilytica(AY950663)

94 JN167688

Firmicutes 3 Paenibacillus CHB40 1 1.11 Paenibacillus daejeonensis(AF391124)

99 JN167679

CHB15 1 1.11 Paenibacillus xylanilyticus(AY427832)

99 JN167687

Sediminibacillus CHB5 1 1.11 Sediminibacillus halophilus(AM905297)

98 JN167689

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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

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

Group No. of

OTUs

Genus Strain No. of

clones

Percentage of

total clones

Closest NCBI match Percentage of

indentity

GenBank

accession no.

Proteobacteria 6 Klebsiella ZC150 87 65.41 Klebsiella variicola(AJ783916)

100 JN167690

ZC138 37 27.82 K. pneumoniae(ACZD01000038)

99 JN167691

Pseudomonas ZC73 4 3.01 Pseudomonasplecoglossicida(AB009457)

98 JN167693

Stenotrophomonas ZC37 3 2.26 S. pavanii (FJ748683) 100 JN167694

Sphingomonas ZC42 1 0.75 Sphingomonasechinoides (AJ012461)

99 JN167695

Rhizobium ZC25 1 0.75 Rhizobium massiliae(AF531767)

100 JN167696

Table 2 continued

Group No. of

OTUs

Genus Strain No. of

clones

Percentage of

total clones

Closest NCBI match Percentage

of indentity

GenBank

accession no.

Actinobacteria 3 Corynebacterium CHB77 2 2.22 Corynebacteriumpseudogenitalium(ABYQ01000237)

98 JN167659

Nocardia CHB72 1 1.11 Nocardia soli (AF430051) 99 JN167670

Lentzea CHB50 1 1.11 Lentzea flaviverrucosa(AF183957)

100 JN167672

Bacteroidetes 3 Flavobacterium CHB145 1 1.11 Flavobacterium degerlachei(AJ557886)

98 JN167656

CHB82 1 1.11 Flavobacterium aquatile(M62797)

97 JN167669

Chryseobacterium CHB47 1 1.11 Chryseobacterium hominis(AM261868)

99 JN167678

Uncultured

bacteria

2 – CHB119 2 2.22 Uncultured bacterium

(EU335174)

96 JN167667

– CHB29 1 1.11 Uncultured bacterium

(EU276548)

95 JN167681

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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

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

Group No. of

OTUs

Genus Strain No. of

clones

Percentage of

total clones

Closest NCBI match Percentage of

indentity

GenBank

accession no.

Proteobacteria 11 Serratia J24D34 87 68.50 S. marcescens(AB061685)

99 JN167697

Sphingomonas J24D36 21 16.54 S. echinoides(AJ012461)

99 JN167698

J24D56 2 1.57 Sphingomonasdokdonensis(DQ178975)

99 JN167701

Pantoea J24D42 1 0.79 P. dispersa (DQ504305) 99 JN167699

Luteibacter J24D54 4 3.15 Luteibacter anthropi(FM212561)

100 JN167700

Burkholderia J24D61 1 0.79 Burkholderia gladioli(EU024168)

100 JN167702

Leclercia J24D13 4 3.15 L. adecarboxylata(AB273740)

99 JN167703

Tepidimonas J24D72 1 0.79 Tepidimonas aquatic(AY324139)

99 JN167705

Tatumella J24D131 1 0.79 T. morbirosei(EU344769)

99 JN167706

Enterobacter J24D143 1 0.79 E. cancerogenus(Z96078)

99 JN167707

Thermomonas J24D120 1 0.79 Thermomonas brevis(AJ519989)

97 JN167708

Firmicutes 1 Lactobacillus J24D23 3 2.36 Lactobacillus iners(ACLN01000018)

99 JN167704

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

Group No. of

OTUs

Genus Strain No. of

clones

Percentage of

total clones

Closest NCBI

match

Percentage of

indentity

GenBank

accession no.

Proteobacteria 2 Pantoea J89E143 118 99.16 P. dispersa(DQ504305)

100 JN167709

Sphingomonas J89E132 1 0.84 S. echinoides(AJ012461)

99 JN167710

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

Group No. of

OTUs

Genus Strain No. of

clones

Percentage of

total clones

Closest NCBI

match

Percentage of

indentity

GenBank

accession no.

Proteobacteria 3 Pantoea YYIb34 34 24.46 P. dispersa(DQ504305)

100 JN167784

YYI105 68 48.92 P. agglomerans(AJ233423)

99 JN167785

Sphingomonas YYI146 37 26.62 S. echinoides(AJ012461)

99 JN167786

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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

Table 7 Distribution of 16S rRNA clones detected from endophytes of Zhengdan 958

Group No. of

OTUs

Genus Strain No. of

clones

Percentage

of total

clones

Closest NCBI match Percentage

of indentity

GenBank

accession no.

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

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(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

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

Group No. ofOTUs

Genus Strain No. ofclones

Percentage oftotal clones

Closest NCBI match Percentageof indentity

GenBankaccession no.

Proteobacteria 30 Brevundimonas JDGb36 1 0.99 Brevundimonas diminuta(GL883089)

99 JN167744

JDG95 1 0.99 Brevundimonas naejangsanensis(FJ544245)

99 JN167764

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(DQ298025)

99 JN167749

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(AB175642)

99 JN167779

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(AB267476)

98 JN167781

Sphingobacterium JDG129 1 0.99 Sphingobacterium daejeonense(AB249372)

99 JN167759

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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,

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

Group No. of

OTUs

Genus Strain No. of

clones

Percentage of

total clones

Closest NCBI match Percentage of

indentity

GenBank

accession no.

Proteobacteria 2 Leclercia JYH3 96 73.85 L. adecarboxylata(AB273740)

99 JN167782

Enterobacter JYH4 34 26.15 E. cancerogenus(Z96078)

99 JN167783

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

dominant

genera

Leclercia(50.00 %)

Roseateles(36.67 %)

Klebsiella(93.23 %)

Serratia(68.50 %)

Pantoea(99.16 %)

Sphingomonas(41.67 %)

Roseateles(31.68 %)

Leclercia(73.85 %)

Pantoea(73.38 %)

The second

dominant

genera

Tatumella(21.09 %)

Acinetobacter(15.56 %)

Pseudomonas(3.01 %)

Sphingomonas(18.11 %)

Sphingomonas(0.84 %)

Bacillus/

Acinetobacter(9.26 %)

Pseudomonas(12.87 %)

Enterobacter(26.15 %)

Sphingomonas(26.62 %)

The third

dominant

genera

Enterobacter(13.28 %)

Burkholderia(10.00 %)

Stenotrophomonas(2.26 %)

Luteibacter/

Leclercia(3.15 %)

– Leclercia(7.41 %)

Sphingomonas(9.90 %)

– –

Table 11 Agronomic traits of four maize hybrids

Seed Agronomic traits

Crude

protein (%)

Crude

starch (%)

Crude fat

(%)

Lysine

(%)

Yuyu 23 10.00 73.12 4.55 0.28 Widely adaptable, high yielding, high quality, pathogen resistant and highly

fecund

Zhengdan

958

9.33 73.02 3.98 0.25 High and stable yields, good quality, pathogen resistance, good fecundity and

good drought and heat tolerance

Jingdan

28

9.47 74.82 4.01 0.25 Good disease and lodging resistance and green-keeping capability

Jingyu 11 8.17 75.32 4.17 0.26 High yielding, high lodging resistance, disease resistance and shade and high

moisture tolerance

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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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