Study on Diversity of Endophytic Bacterial Communities in Seeds of Hybrid Maize and Their Parental Lines

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

Archives of Microbiology

ISSN 0302-8933 Volume 194 Number 12

Arch Microbiol (2012) 194:1001-1012 DOI 10.1007/s00203-012-0836-8

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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 bacterium Acinetobacter (9.26 %) was also the second beneficial bacteria and pathogens. Using the non-culture dominant bacterium of its male parent. In the hybrid methods of building 16S rDNA libraries, we investigated Jingdan 28, the second dominant bacterium Pseudomonas the endophytic bacterial communities of seeds of four (12.78 %) was also the second dominant bacterium of its hybrid maize offspring and their respective parents. The female parent, and its third dominant bacterium Sphingo- results of this study show that the hybrid offspring Yuyu monas (9.90 %) was the second dominant bacterium of its 23, Zhengdan958, Jingdan 28 and Jingyu 11 had 3, 33, 38 male parent and detected in its female parent. In the hybrid and 2 OTUs of bacteria, respectively. The parents Ye 478, Jingyu 11, the first dominant bacterium Leclercia Chang 7-2, Zheng 58, Jing 24 and Jing 89 had 12, 36, 6, 12 (73.85 %) was the third dominant bacterium of its male and 2 OTUs, respectively. In the hybrid Yuyu 23, the parent, and the second dominant bacterium Enterobacter dominant bacterium Pantoea (73.38 %) was detected in its (26.15 %) was detected in its male parent. As far as we female parent Ye 478, and the second dominant bacterium know, this was the first research reported in China on the of Sphingomonas (26.62 %) was detected in both its female diversity of the endophytic bacterial communities of the (Ye 478) and male (Chang 7-2) parent. In the hybrid seeds of various maize hybrids with different genotypes. Zhengdan 958, the first dominant bacterium Stenotropho- monas (41.67 %) was detected in both the female (Zheng Keywords Hybrid maize Seed endophytic bacteria 58) and male (Chang 7-2) parent. The second dominant Bacterial diversity Culture-independent method

Abbreviation Communicated by Ursula Priefer. CTAB Cetyltrimethylammonium bromide

Y. Liu S. Zuo Y. Zou W. Song (&) College of Life Sciences, Capital Normal University, Beijing 100048, People’s Republic of China e-mail: [email protected] Introduction

Y. Liu China National Research Institute of Food and Fermentation Endophytic bacteria are a class of endosymbiotic micro- Industries, Beijing 100027, People’s Republic of China organisms that were able to colonize and healthfully coexist with plant tissues (Kloepper and Beauchamp 1992). Y. Liu Seeds act as the continuation organ of plants and serve as China Center of Industrial Culture Collection, China National Research Institute of Food and Fermentation an important means of agriculture production (Guan 2009), Industries, Beijing 100027, People’s Republic of China but can also carry a variety of pathogens and beneﬁcial bacteria. Many studies have conﬁrmed that the surface and L. Xu interior of seeds bear a variety of microbial organisms Maize Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, (Nelson 2004). During seed germination, growth and People’s Republic of China survival of these endophytic microbial communities and 123 Author's personal copy

1002 Arch Microbiol (2012) 194:1001–1012 the microbial communities from the soil are facilitated Jing 24 and Jing 89) supplied by Professor Jiuran Zhao at (Bacilio-Jimeńe et al. 2001; Cottyn et al. 2001). The Beijing Academy of Agriculture and Forestry Sciences. The microbes then may facilitate and interact with surrounding genetic relationships among the samples are shown in Fig. 1. plants, significantly impacting soil fertility and plant All the samples were collected in March 2011 from the growth (Barea et al. 2005). Beijing Academy of Agriculture and Forestry Sciences Early studies have shown that the genotypes of plants experimental plot in Sanya, Hainan (18.35774333340131 N, can impact the microorganisms coexisting with the plants. 109.18169975280762 E, southern China) and stored at 4 °C. Michiels et al. (1989) found that the genotype of a plant Maize seeds were washed with sterile water and controls the composition and the quantity of root exudates immersed in 70 % ethanol for 3 min. They were then and correlates with the quantity and activity of bacteria washed with fresh sodium hypochlorite solution (2.5 % colonizing the rhizosphere (e.g., azotobacter). Neal et al. available Cl-) for 5 min, rinsed with 70 % alcohol for 30 s (1973) studied the species of microorganisms found in the and finally washed 5–7 more times with sterile water (Sun rhizosphere of different wheat genotypes and found that the et al. 2008). The aliquots of the final rinsing water were rhizosphere of mutant wheat contained different species spread on Luria–Bertani solid medium plates and cultured than the wild types. They suggested that the genotype of for 3 days at 28 °C in order to confirm that the seeds were the plants determined the species of microorganisms col- sterilized and no seed surface bacteria remained. Only the onizing the rhizosphere. seed samples that were confirmed as sterile were used for Though there are many studies on rhizosphere microor- subsequent analysis. ganism communities, up to now, few studies have focused on microorganisms associated with seeds (Cankar et al. DNA extraction and PCR amplification of the bacterial 2005) and even fewer have attempted to correlate endo- 16S rRNA gene phytic bacteria of maize seeds with their genotypes. In order to understand the structure of endophytic bacterial com- About 5.0 g of surface-sterilized maize seeds was frozen munities of different seed genotypes and explore the rela- with liquid nitrogen and quickly ground into a fine powder tionship of the endophytic bacterial community structures with a precooled sterile mortar. Then, CTAB procedure of seeds of the filial generation of maize hybrids and their was used to extract the seed and bacterial DNA of all parental lines, we constructed 16S rDNA libraries to iden- samples (Sun et al. 2008). The DNA was then resuspended tify the endophytic bacteria colonizing four combination in 30 lL sterile Milli-Q water. offspring of hybrid maize seeds and their respective parents. The 16S rDNA of the indigenous bacteria within the seeds was amplified using 799f (50-AACAGGATTAGATA CCCTG-30) and 1492r (50-GGTTACCTTGTTACGACTT-30) Materials and methods as primers. These 2 primers were chosen because they can separate bacterial and maize mitochondrial products (Sun Maize seed sampling and surface sterilization et al. 2008). The 50-lL PCR reaction mixture contained 50 ng of DNA extract, 1 9 Taq reaction buffer, 20 pmol of Seeds were collected from four hybrid combinations of each primer, 200 lmol of dNTP and 1.5 units Taq enzyme maize (Zea mays L.) (Yuyu 23, Zhengdan958, Jingdan 28, (Ferments). Reaction procedure: initial denaturation at Jingyu 11) and their parents (Ye 478, Chang 7-2, Zheng 58, 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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Arch Microbiol (2012) 194:1001–1012 1003 annealing at 52 °C for 1 min, elongation at 72 °C for those of the two parents. Yuyu 23 seeds had only 2 bacterial 1 min, after 30 circulations, extension at 72 °C for 10 min. OTUs, and the dominant bacteria genera were Pantoea The temperature was then decreased to 52 °C to allow (73.38 %) and Sphingomonas (26.62 %). The male and annealing for 1 min then increased to 72 °C for 1 min of female parent seeds, containing 12 and 36 OTUs, respec- elongation. After 30 cycles of the above, the temperature tively were richer in endophytic bacterial species than their was held at 72 °C for 10 min for extension. The PCR offspring. For the female parent, Ye 478, the dominant products were then electrophoretically separated. The band bacteria genera were Leclercia (50.00 %), Tatumella at approximately 750 bp was excised and purified using the (21.09 %) and Enterobacter (13.28 %), while those of the Wizard SV Gel and PCR Clean-up System (Promega) as male parent, Chang 7-2, were Roseateles (36.67 %), Aci- described by the manufacturer. netobacter (15.56 %) and Burkholderia (10.00 %). Though the first (Pantoea) and second (Sphingomonas) dominant Construction of the16S rRNA gene clone library bacterial genera of the offspring was not dominant in either of the parents, Pantoea was detected in the female parent, The purified PCR products were ligated into the T3 vector and Sphingomonas was detected in both the female and according to the protocol supplied by the manufacturer male parent (Tables 1, 2, 6, 10). (Transgen, China). Escherichia coli DH5a competent cells In Zhengdan 958, the hybrid combination of Zheng (Transgen, China) were transformed with the ligation 58 9 Chang 7-2, the seeds had more species of endophytic products and spread onto LB agar plates with ampicillin bacteria than and some similarities in dominant bacteria (100 lg/ml) and X-gal/IPTG on the surface for standard with the parents. Zhengdan 958 seeds contained 33 OTUs, blue and white screening. White colonies were randomly while the female and male parent seeds contained 7 and 36 picked and cultured in liquid LB over night. OTUs, respectively. The dominant bacteria of Zhengdan 958 seeds were Sphingomonas (41.67 %), Bacillus/Acine- Sequencing and phylogenetic analysis tobacter (9.26 %) and Leclercia (7.41 %). The dominant bacteria genera in the female parent, Zheng 58, were Sequencing was performed on 100–150 randomly chosen Klebsiella (93.23 %), Pseudomonas (3.01 %) and Steno- clones. Partial sequences of cloned 16S rRNA genes were trophomonas (2.26 %), while those of male parent, Chang sequenced with an ABI 3730 DNA sequencer (ABI, USA). 7-2, were Roseateles (36.67 %), Acinetobacter (15.56 %) All of the nucleotide sequences, approximately 700 bases, and Burkholderia (10.00 %). In this combination, the sec- were either compared with the NCBI database using ond dominant genus (Acinetobacter) of the hybrid off- BLASTN or aligned by the identification analysis of spring was consistent with the second dominant bacteria of EzTaxon server 2.1 (Chun et al. 2007). Sequences with its male parent, and the first dominant bacterium (Steno- [97 % similarity were assigned to the same species. 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 Results offspring, Jingdan 28. Jingdan 28 had more species of endophytic bacteria than its parents and similar dominant Electrophoresis of the plant seed and the endophytic bacteria genera to its parents. The offspring seeds of Jingdan 28 ligation products showed two bands. One lied between 1,000 contained 38 OTUs, compared to 7 and 12 OTUs for the and 1,500 bp, which corresponds with maize mitochondrial female and male parent, respectively. For Jingdan 28, the 18S rDNA; the other was found between 700 and 800 bp, dominant bacteria genera were Roseateles (31.68 %), corresponding with endophytic bacteria 16S rDNA. We Pseudomonas (12.87 %) and Sphingomonas (9.90 %). As amplified the target fragments of 100–150 clones from each for the female parent, Zheng 58, the dominant bacteria library. The sequence information was submitted to the genera were Klebsiella (93.23 %), Pseudomonas (3.01 %) GenBank accession (accession No. JN167639–JN167786, and Stenotrophomonas (2.26 %), while those of the male excluding JN167649, JN167668, JN167692 and JN167673). parent, Jing 24, were Serratia (68.50 %), Sphingomonas The combination offspring of hybrid maize, Yuyu 23, (18.11 %) and Luteibacter/Leclercia (3.15 %). In this Zhengdan958, Jingdan 28 and Jingyu 11 had 3, 33, 38 and combination, the second dominant bacterium (Pseudomo- 2 OTUs, respectively. The parents Ye 478, Chang 7-2, nas) of Jingdan 28 was consistent with the second domi- Zheng 58, Jing 24 and Jing 89 had 12, 36, 6, 12 and 2 nant bacterium of its female parent, and the hybrid OTUs, respectively (Tables 1, 2, 3, 4,5, 6, 7, 8, 9). offspring’s third dominant bacterium (Sphingomonas) was In the Yuyu 23, the hybrid offspring of Ye 478 9 Chang consistent with the second dominant bacterium of its male 7-2, the endophytic bacterial community was less species parent. Furthermore, all of Jingdan 28’s dominant bacteria rich than and the dominant bacteria were inconsistent with were detected in its female parent (Tables 3, 4, 8, 10). 123 Author's personal copy

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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. Acinetobacter, and Serratia were present in both offspring The offspring seed of Jingyu 11 had fewer species of (Tables 3, 7, 8). endophytic bacteria, but similar dominant bacterium gen- The hybrid offspring Jingdan 28 and Jingyu 11 both had era relative to its parents. Jing 11 seeds contained only 2 Jing 24 as their male parent. There were clear differences OTUs. The female and male parent seeds’ endophytic between the endophytic bacteria of these 2 hybrids. There bacteria were made up of 2 and 12 OTUs, respectively. For were no similarities in the dominant endophytic genera of Jingyu 11, the dominant bacteria genera were Leclercia the 2 hybrids, but the dominant endophytic bacterium (73.85 %) and Enterobacter (26.15 %). In the female (Sphingomonas) of Jingdan28 and the dominant endophytic parent, Jing 89, the dominant bacteria genera were Pantoea bacterium of Jingyu 11 were both detected in the male (99.16 %) and Sphingomonas (0.84 %). In the male parent, parent (Tables 4, 8, 9, 10). Jing 24, the dominant bacteria genera were Serratia There were obvious differences among the four hybrids (68.50 %), Sphingomonas (18.11 %) and Luteibacter/ and their male parent seeds in number and species of Leclercia (3.15 %). In this combination, the first dominant endophytic bacteria, but most dominant endophytic bacte- bacterium (Leclercia) of the hybrid offspring was con- ria of the offspring were also detected in their parental sistent with the third dominant bacterium of the male seeds. When comparing the endophytic bacteria, especially parent, and the second dominant bacterium (Enterobacter) the dominant endophytic species found in hybrids with of the offspring was detected in the male parent (Tables 4, genetic correlations have some relevance with their parents 5, 9, 10). (Tables 1, 2, 3, 4, 5, 6, 7, 8, 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 Discussion endophytic bacteria species. Sphingomonas was the dominant endophytic bacterial genus for both offspring, and it Seeds are carriers of both microphyte parental genes (Guan was detected in the male parent. The genus, Pantoea was 2009) and a variety of beneficial bacteria and pathogens. also detected in both Yuyu 23 and Zhengdan 958 These microorganisms originate from various microbial (Tables 2, 6, 7). communities born on the seed surface (Nelson 2004). To The hybrid offspring of Zhengdan 958 and Jingdan 28 the best of our knowledge, studies on the endophytic bac- had the same female parent (Zheng 58), but with different teria of maize are much more less than rice. Using culture male parents. Both offspring had more endophytic bacteria methods, Rijavec et al. (2007) identified the endophytic species than their parents. Sphingomonas, which was also bacteria genera released during germination of four types present in the female parent, was the dominant endophytic of maize seeds. Johnston-Monje and Raizada (2011) found bacterial genus for both Zhengdan 958 and Jingdan 28. that seed endophyte community composition varied in The bacterial genera Shigella, Burkholderia, Acidovorax, 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 different tomato genotypes. Adams and Kloepper (2002) seeds across boundaries of evolution, ethnography and investigated the impact of cotton plant genotype on the ecology. This study is an attempt to use non-culture inherent bacterial population of their seeds, seedling stems methods to study the diversity of endophytic bacterial and root tissues. They found that cotton plants have communities associated with the seeds of the new type the capacity to carry endophytic bacterial communities maize hybrids and their parents which autonomously cul- immediately after seed germination and that in the process tured by China. The purpose of the research is to investi- of seed germination and seedling development, the distinct gate the influence of maize seed genotype on the structure genetic, morphological and physiological characteristics of of seed endophytic bacterial communities. individual cotton cultivars led to differences in the endo- The results show clear differences in number and spe- phytic bacterial community structure among the cultivars. cies of endophytic bacteria among the seeds of the four Picard and Bosco (2006) found that the filial generation of offspring hybrids and their parents. One of the reasons for hybrid maize contains more protein than their parents, these differences may be variability in genotype among the which results in attraction of more pseudomonas to the plants investigated. Simon et al. (2001) found the growth of filial generation roots. Xiao et al. (1995) reported that inherent and inoculated bacteria differed on the seeds of 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 nutritional structure among the genotypes of maize seeds superior to their parents. In the present study, all offspring are probably one of the key reasons for differences in of the four maize hybrids had strong agronomic traits endophytic bacterial communities. (Table 11) (Sun et al. 2005; San et al. 2007; Chen et al. It should be noted that the factors that affect the endo- 2009). Hence, we can infer that the differences in 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, genetically related hybrid maize. It was found that most but also on the seed itself. Other important variables dominant endophytic of hybrid offspring were detected in influencing the bacterial communities of seeds and their the parental seeds, indicating that there is a continuity of succession may be seed shape, different parts of tissue and endophytic bacteria from the parental to the offspring seed. development and germination stage (Zou et al. 2011). Reports have shown that endophytic bacteria colonize plant The above results also show that the dominant endo- seeds and become an important source of endophytic bac- phytic bacteria of the seeds of the four hybrid maize off- teria for the growing plants (Feng and Song 2001). Recently, spring for this study were also detected in parental seeds. we found that the bacteria in the hybrid seeds with the Additionally, the endophytic bacterial communities in the contents more than 5 % could also be detected in their male seeds of genetically related offspring had similar species or female parental seeds. Endophytic bacterial communities and, in particular, dominant genera compositions. We have of offspring seeds are likely affected by the parental strains illustrated two possible reasons for these trends: (1) The (results unpublished). The structures of endophytic bacterial combined hybrid maize seeds are genetically related; (2) in communities of genetically related offspring, namely hybrid this study, all seed samples were collected at the same period offspring with the same father or the same mother, have in the same experimental field, so the environmental factors some similarities due to their common parent strains. that may influence bacterial communities were relatively Soil environment in which a plant grows is an important uniform. The present study selected a combination of 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 On the other hand, certain genes of plants and bacteria bacteria detected in this study were common soil bacteria. determine the interaction between the two and determine There are two factors that bacteria in the soil environment whether the bacteria are colonized in the plant or not. The become the source of endophytic bacteria of plants. On the unique characteristics of different types of plants will allow one hand, the roots of plants are exposed to bacteria in the different species of bacteria to colonize their body or body soil environment during growth and development, which surface (Hardoim et al. 2008). Physiological characteristics provides opportunities for bacteria to enter into the plants. 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 dispersa identified in this study produces an esterase that present study found that the dominant bacteria genera (i.e., irreversibly degrades pathogenic substance and thus Sphingomonas, Leclercia, Pantoea, Pseudomonas, Acine- restricts the growth of plant pathogens (Zhang and Birch tobacter, Roseateles and Enterobacter) of maize seeds 1997). Pantoea agglomerans are able to fix nitrogen and grown in a relatively uniform environment tend to be found secreted hormone of the plant (Feng et al. 2003). Serratia in relative abundance in that environment, these species marcescens are able to produce antibiotics providing pro- could be more adaptable to the unique physiological tection against some plant pathogens (Wei et al. 1996). environment within the mature maize seeds of the hybrid Klebsiella pneumoniae NG14, which has been isolated type they were found in. This physiological environment is from rice root, appears to promote plant growth through determined by the species and genotype of the seed. synthesis of auxin IAA from indole-3-pyruvate, biological Among the detected dominant bacteria genera (Pseu- nitrogen fixation and colonization of the root surface and domonas, Acinetobacter, Bacillus, Sphingomonas, Entero- root vascular tissue within the cavity of the rice seedlings bacter, Burkholderia and Klebsiella), some can be plant (Liu et al. 2011). These plant growth-promoting bacteria growth-promoting bacteria (Sturz 1995; Videira et al. can be used to increase yield, improve quality, enhance 2009; Lucy et al. 2004; Liu et al. 2011), which may directly pathogen resistance and shortened growth cycles through or indirectly affect the growth and development of plants biological nitrogen fixation, secretion of plant growth (Feng and Song 2001). The dominant bacterium Pantoea regulators and production of antibiotics. At the same time,

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Arch Microbiol (2012) 194:1001–1012 1011 the beneficial effects of these growth-promoting endo- Hardoim PR, van Overbeek LS, van Elsas JD (2008) Properties of phytic bacteria on plants likely improve the environmental bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16:463–471 conditions within the plant for the same bacteria causing a Jefferey SB, Daniel PR, Estelle RC (1999) Microbial community positive feedback loop. structure and function in the spermosphere as affected by soil This study is the first attempt to employ culture-inde- and seed type. Can J Microbiol 45:138–144 pendent techniques to investigate the endophytic bacteria of Johnston-Monje D, Raizada MN (2011) Conservation and diversity of seed associated endophytes in Zea across boundaries of evolu- the seeds of hybrid maize offspring and their parents. Our tion, ethnography and ecology. PLoS ONE 6(6):e20396. doi: results provide a solid foundation for further study of the 10.1371/journal.pone.0020396 correlation of endophytic bacteria community with structure Kloepper JW, Beauchamp CJ (1992) A review of issues related to seed genotypes. This provides the necessary baseline infor- measuring colonization of plant roots by bacteria. Can J Microbiol 38:1219–1232 mation for further studies of the relationships and interac- Liu Y, Wang H, Sun XL, Yang HL, Wang YS, Song W (2011) tions between endophytic bacteria and their host seeds and Study on mechanisms of colonization of nitrogen-fixing may provide a new starting point for investigations of plant PGPB, Klebsiella pneumoniae NG14 on the root surface of heterosis. Further research on correlations among the rice and the formation of biofilm. Curr Microbiol 62:1113– 1122 endophytic bacteria communities of different seed geno- Lucy M, Reed E, Glick BR (2004) Applications of free living plant types and seeds of hybrid offspring and their parents and the growth-promoting rhizobacteria. Antonie Van Leeuwenhoek agronomic traits of these bacteria is necessary. 86:1–25 Michiels K, Vanderleyden J, Vangool A (1989) Azospirillum—Plant Acknowledgments This work was supported by the National Natural Root Associations—a review. Biol Fert Soils 8:356–368 Science Foundation of China (No. 30770069), the Science Foundation of Neal JL, Larson RI, Atkinson TG (1973) Changes in rhizosphere Beijing (No. 5092004). National Science and Technology Support Plans populations of selected physiological groups of bacteria related of China (2012BAK17B11) and International Science and Technology to substitution of specific pairs of chromosomes in spring wheat. Cooperation Projects of Beijing (Z111105054611011). 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