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

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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 1 23 Your article is protected by copyright and all rights are held exclusively by Springer- Verlag. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication. 1 23 Author's personal copy 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 Á Seed endophytic bacteria Á 58) and male (Chang 7-2) parent. The second dominant Bacterial diversity Á Culture-independent method Abbreviation Communicated by Ursula Priefer. CTAB Cetyltrimethylammonium bromide Y. Liu Á S. Zuo Á Y. Zou Á 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-Jime´ne 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 123 Author's personal copy 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).
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