Endophytic bacteria in Miscanthus seed: implications for germination, vertical inheritance of endophytes, plant evolution and breeding

Abstract

With growing interest in the role of microbiomes, and symbionts in particular, the aim of this study was to determine the diversity of the bacterial endophyte population within Miscanthus and to ascertain the extent of vertical transmission via the seed. A great diversity of endophytic bacteria was found in all parts of the mature plant (rhizome, root, stem and leaf), and in seedlings grown from sterilized seed grown in sterile conditions. A total of three phyla and five families of bacteria were identified as cultures compared to 19 phyla and 85 families using 16S rDNA amplification and sequencing. Not all cultured bacteria could be identified by 16S rDNA, implying that the true diversity is even greater. More bacterial diversity was identified in sterile-grown seedlings than in all parts of the mature plant combined, 17 and 13 phyla, respectively, with 11 in common. Five phyla were present in all plant samples examined. Vertical transmission via the seed may therefore be a major source of endophytes in Miscanthus, presumably supplemented by ingress of soil bacteria as the plant grows. Bacteria identified from the mature plant were predominantly similar to known bacterial sequences in GenBank, but a small number from the stem and many from the seed were novel, potentially adapted to an in planta life cycle. Endophytic bacteria were found to form spores and other dense structures, and this provides a mechanism for long-term survival and seed transmission. The staining of germinating seeds identified bacteria at the root tip of the emerging radicle. We propose that seed transmission of bacterial endophytes requires adaptation of both plant and microbes, plays a role in germination and has evolutionary significance and implications for future plant breeding approaches, in Miscanthus and more widely.

References Adler CJ, Dobney K, Weyrich LS et al. (2013) Sequencing ancient calcified dental plaque shows changes in oral microbiota with dietary shifts of the Neolithic and Industrial revolutions. Nature Genetics, 45, 450–455. Amman RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews, 59, 143–169. Baldani JI, Baldani VLD, Seldin L, Dobereiner J (1986) Characterization of Herbaspirillum seropedicae gen. nov., sp. nov., a root-associated nitrogen-fixing bacterium. International Journal of Systematic Bacteriology, 36, 86–93. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2008) BLAST+: architecture and applications. BMC Bioinformatics, 10, 421. Cankar K, Kraigher H, Ravnikar M, Rupnik M (2005) Bacterial endophytes from seeds of Norway spruce (Picea abies L. Karst). FEMS Microbiology Letters, 244, 341–345. Cock PJ, Antao T, Chang JT et al. (2009) Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics, 25, 1422–1423. Compant S, Kaplan H, Sessitsch A, Nowak J, Barka EA, Clément C (2008) Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: from the rhizosphere to inflorescence tissues. FEMS Microbiology Ecology, 63, 84–93. Compant S, Clément C, Sessitsch A (2010) Plant growth-promoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biology and Biochemistry, 42, 669–678. Compant S, Mitter B, Colli-Mull JG, Gangl H, Sessitsch A (2011) Endophytes of grapevine flowers, berries, and seeds: identification of cultivable bacteria, comparison with other plant parts, and visualization of niches of colonization. Microbial Ecology, 62, 188–197. DeAngelis KM, Brodie EL, DeSantis TZ, Andersen GL, Lindow SE, Firestone MK (2008) Selective progressive response of soil microbial community to wild oat roots. The ISME Journal, 3, 168–178. Dichosa AEK, Daughton AR, Reitenga KG, Fitzsimons MS, Han CS (2014) Capturing and cultivating single bacterial cells in gel microdroplets to obtain near-complete genomes. Nature Protocols, 9, 608–621. Eckert B, Weber OB, Kirchhof G, Halbritter A, Stoffels M, Hartmann A (2001) Azospirillum doebereinerae sp. nov., a nitrogen-fixing bacterium associated with the C4-grass Miscanthus. International Journal of Systematic and Evolutionary Microbiology, 51(Pt 1), 17–26. Elbeltagy A, Nishioka K, Sato T et al. (2001) Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species. Applied and Environmental Microbiology, 67, 5285–5293. Evans AGL, Davey HM, Cookson A, Currinn H, Cooke-Fox G, Stanczyk PJ, Whitworth DE (2012) Predatory activity of Myxococcus xanthus outer-membrane vesicles and properties of their hydrolase cargo. Microbiology (Reading, England), 158(Pt 11), 2742–2752. Ewald PW (1987) Transmission modes and evolution of the parasitism-mutualism continuum. Annals of the New York Academy of Sciences, 503, 295–306. Farrar K, Bryant D, Cope-Selby N (2014) Understanding and engineering beneficial plant-microbe interactions: plant growth promotion in energy crops. Plant Biotechnology Journal, 12, 1193–1206. Gagne-Bourgue F, Aliferis KA, Seguin P, Rani M, Samson R, Jabaji S (2013) Isolation and characterization of indigenous endophytic bacteria associated with leaves of switchgrass (Panicum virgatum L.) cultivars. Journal of Applied Microbiology, 114, 836–853. Gillis M, Kersters K, Hoste B et al. (1989) Acetobacter diazotrophicus sp. nov., a nitrogen-fixing acetic acid bacterium associated with sugarcane. International Journal of Systematic Bacteriology, 39, 361–364. Hardoim PR, van Overbeek LS, van Elsas JD (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends in Microbiology, 16, 463–471. Hardoim PR, Hardoim CC, Van Overbeek LS, Van Elsas JD (2012) Dynamics of seed-borne rice endophytes on early plant growth stages. PLoS ONE, 7, e30438. Heaton EA, Dohleman FG, Long SP (2008) Meeting US biofuel goals with less land: the potential of Miscanthus. Global Change Biology, 14, 2000–2014. Islam SMA, Math RK, Kim JM et al. (2010) Effect of plant age on endophytic bacterial diversity of balloon flower (Platycodon grandiflorum) root and their antimicrobial activities. Current Microbiology, 61, 346–356. James EK (2000) Nitrogen fixation in endophytic and associative symbiosis. Field Crops Research, 65, 197–209. James EK, Gyaneshwar P, Mathan N et al. (2002) Infection and colonization of rice seedlings by the plant growth-promoting bacterium Herbaspirillum seropedicae Z67. Molecular Plant-Microbe Interactions, 15, 894–906. Jensen E, Farrar K, Thomas-Jones S, Hastings A, Donnison I, Clifton-Brown J (2011) Characterization of flowering time diversity in Miscanthus species. GCB Bioenergy, 3, 387–400. Johnston-Monje D, Raizada MN (2011) Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology. PLoS ONE, 6, e20396. Kirchhof G, Reis VM, Baldani JI, Eckert B, Döbereiner J, Hartmann A (1997) Occurrence, physiological and molecular analysis of endophytic diazotrophic bacteria in gramineous energy plants. Plant and Soil, 194, 45-55. Kirchhof G, Eckert B, Stoffels M, Baldani J, Reis V, Hartmann A (2001) Herbaspirillum frisingense sp. nov., a new nitrogen-fixing bacterial species that occurs in C4-fibre plants. International Journal of Systematic and Evolutionary Microbiology, 51, 157–168. Lee S, Flores-Encarnación M, Contreras-Zentella M, Garcia-Flores L, Escamilla JE, Kennedy C (2004) Indole-3-acetic acid biosynthesis is deficient in Gluconacetobacter diazotrophicus strains with mutations in cytochrome c biogenesis genes. Journal of Bacteriology, 186, 5384–5391. Lewandowski I, Clifton-Brown JC, Scurlock JMO, Huisman W (2000) Miscanthus: European experience with a novel energy crop. Biomass and Bioenergy, 19, 209–227. Lewandowski I, Scurlock JMO, Lindvall E, Christou M (2003) The development and current status of perennial rhizomatous grasses as energy crops in the US and Europe. Biomass and Bioenergy, 25, 335–361. Liu Y, Zuo S, Zou Y, Wang J, Song W (2013) Investigation on diversity and population succession dynamics of endophytic bacteria from seeds of maize (Zea mays L., Nongda108) at different growth stages. Annals of Microbiology, 63, 71–79. Loiret FG, Ortega E, Kleiner D, Ortega-Rodés P, Rodés R, Dong Z (2004) A putative new endophytic nitrogen-fixing bacterium Pantoea sp. from sugarcane. Journal of Applied Microbiology, 97, 504–511. Loiret FG, Grimm B, Hajirezaei MR, Kleiner D, Ortega E (2009) Inoculation of sugarcane with Pantoea sp. increases amino acid contents in shoot tissues; serine, alanine, glutamine and asparagine permit concomitantly ammonium excretion and nitrogenase activity of the bacterium. Journal of Plant Physiology, 166, 1152–1161. Miyamoto T, Kawahara M, Minamisawa K (2004) Novel endophytic nitrogen-fixing clostridia from the grass Miscanthus sinensis as revealed by terminal restriction fragment length polymorphism analysis. Applied and Environmental Microbiology, 70, 6580–6586. Monteiro RA, Balsanelli E, Wassem R et al. (2012) Herbaspirillum-plant interactions: microscopical, histological and molecular aspects. Plant and Soil, 356, 175–196. Muthukumarasamy R, Govindarajan M, Vadivelu M, Revathi G (2006) N-fertilizer saving by the inoculation of Gluconacetobacter diazotrophicus and Herbaspirillum sp. in micropropagated sugarcane plants. Microbiological Research, 161, 238–245. Nogales B, Moore ER, Llobet-Brossa E, Rossello-Mora R, Amann R, Timmis KN (2001) Combined use of 16S ribosomal DNA and 16S rRNA to study the bacterial community of polychlorinated biphenyl-polluted soil. Applied and Environmental Microbiology, 67, 1874–1884. Okunishi S, Sako K, Mano H, Imamura A, Morisaki H (2005) Bacterial flora of endophytes in the maturing seed of cultivated rice (Oryza sativa). Microbes and Environments, 20, 168–177. Olivares FL, Baldani VLD, Reis VM, Baldani JI, Dobereiner J (1996) Occurrence of the endophytic diazotrophs Herbaspirillum spp. in roots, stems, and leaves, predominantly of Gramineae. Biology and Fertility of Soils, 21, 197–200. Oliveira ALM, Canuto EL, Urquiaga S, Reis VM, Baldani JI (2006) Yield of micropropagated sugarcane varieties in different soil types following inoculation with diazotrophic bacteria. Plant and Soil, 284, 23–32. Quecine MC, Araújo WL, Rossetto PB et al. (2012) Sugarcane growth promotion by the endophytic bacterium Pantoea agglomerans 33.1. Applied and Environmental Microbiology, 78, 7511–7518. Rinke C, Schwientek P, Sczyrba A et al. (2013) Insights into the phylogeny and coding potential of microbial dark matter. Nature, 499, 431–437. Rosenblueth M, Martínez-Romero E (2006) Bacterial endophytes and their interactions with hosts. Molecular Plant-Microbe Interactions: MPMI, 19, 827–837. Rouws LFM, Meneses CHSG, Guedes HV, Vidal MS, Baldani JI, Schwab S (2010) Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes. Letters in Applied Microbiology, 51, 325–330. Schloss PD, Westcott SL, Ryabin T et al. (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Applied and Environmental Microbiology, 75, 7537–7541. Stewart AD, Logsdon JM, Kelley SE (2005) An empirical study of the evolution of virulence under both horizontal and vertical transmission. Evolution, 59, 730–739. Straub D, Yang H, Liu Y, Tsap T, Ludewig U (2013a) Root ethylene signalling is involved in Miscanthus sinensis growth promotion by the bacterial endophyte Herbaspirillum frisingense GSF30(T). Journal of Experimental Botany, 64, 4603–4615. Straub D, Rothballer M, Hartmann A, Ludewig U (2013b) The genome of the endophytic bacterium H. frisingense GSF30T identifies diverse strategies in the Herbaspirillum genus to interact with plants. Frontiers in Microbiology, 4, 168. Truyens S, Weyens N, Cuypers A, Vangronsveld J (2015) Bacterial seed endophytes: genera, vertical transmission and interaction with plants. Environmental Microbiology Reports, 7, 40–50. Webb KJ, Cookson A, Allison G, Sullivan ML, Winters AL (2013) Gene expression patterns, localization, and substrates of polyphenol oxidase in red clover (Trifolium pratense L.). Journal of Agricultural and Food Chemistry, 61, 7421–7430. Wei C-Y, Lin L, Luo LJ et al. (2014) Endophytic nitrogen-fixing Klebsiella variicola strain DX120E promotes sugarcane growth. Biology and Fertility of Soils, 50, 657–666. Weyens N, van der Lelie D, Taghavi S, Vangronsveld J (2009) Phytoremediation: plant-endophyte partnerships take the challenge. Current Opinion in Biotechnology, 20, 248–254. Yarza P, Yilmaz P, Pruesse E et al. (2014) Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nature Reviews Microbiology, 12, 635–645.