Plant Soil DOI 10.1007/s11104-017-3368-9

COMMENTARY

Commentary: seed bacterial inhabitants and their routes of colonization Carolina Escobar Rodríguez & Birgit Mitter & Matthieu Barret & Angela Sessitsch & Stéphane Compant

Received: 21 March 2017 / Accepted: 3 August 2017 # Springer International Publishing AG 2017

Abstract Background Seeds host bacterial inhabitants but only a limited knowledge is available on which taxa inhabit seed, which niches could be colonized, and what the routes of colonization are. Scope Within this commentary, a discussion is provided on seed bacterial inhabitants, their taxa, and from where derive the seed colonizers. Conclusions Seeds/and grains host specific bacteria deriving from the anthosphere, carposphere, or from cones of gymnosperms and inner tissues of plants after a long colonization from the soil to reproductive organs. Keywords Seed . Colonization . Bacteria

Since Bernheim and Lehmann disputed the existence of bacterial associations with cereal grains in 1889 (reviewed in Smith 1911), it has been acknowledged that seeds are inhabited by several microorganisms with

Responsible Editor: Eric B. Nelson. C. E. Rodríguez : B. Mitter : A. Sessitsch : S. Compant (*) AIT Austrian Institute of Technology GmbH, Center for Health & Bioresources, Bioresources Unit, Konrad-Lorenz Straße 24, 3430 Tulln, Austria e-mail: [email protected] M. Barret INRA, UMR1345 Institut de Recherches en Horticulture et Semences, SFR4207 QUASAV, F-49071 Beaucouzé, France

some of them contributing to plant growth and health, while others display detrimental or neutral effects on their hosts. Seed-borne microbes are known as pioneer colonizers of the emerging plant and thus represent the foundation of the plant microbiota build-up before microbes are taken up from the surrounding soil (Truyens et al. 2015). Seed endophytes might play a minor role in the mature plant microbiota (Truyens et al. 2015), but as primary plant colonizers they could affect germination, early plant vigor, and survival. However, this still requires experimental evidence, as many other aspects in seed endophyte research - e.g. heritability of bacterial endophytes from seed to seed. Furthermore, a better understanding of colonization routes, preferred niches of seed bacterial inhabitants, and colonization kinetics is needed. Seeds harbor several bacterial inhabitants within specific tissues that can potentially be transmitted across generations. For instance, Johnston-Monje and Raizada (2011) identified some conserved taxa in various maize grains collected from wild ancestors and modern varieties, suggesting that some bacterial groups are conserved across generations despite human selection and cross-continental migration. Another recent study reported that up to 45% of the rice grain bacterial endophytic communities might be vertically transmitted from the parental plant to the second generation (Hardoim et al. 2012). However, only little is known about the exact transmission routes of bacterial seed endophytes and few bacteria were proven to colonize plants over successive generations. One well-documented example is related to the

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leaf symbionts BCandidatus Paraburkholderia spp.^ that are transmitted throughout the entire plant cycle of Psychotria spp. without the need of external infection (von Faber 1912; Miller 1990; van Oevelen et al. 2002, 2004). On the other hand, the vertical transmission of microorganisms is not necessarily associated with its heritability over generations. For instance, Mitter et al. (2017) showed that the endophyte Paraburkholderia phytofirmans PsJN, introduced into wheat and maize grains, colonized vegetative tissues of the offspring plants but was not transmitted to the next generation seeds. Altogether these data suggest that heritability of seed endophytes over generations might depend on bacterial-plant genotype interactions. If some bacterial taxa are found across several plant generations, then it could be assumed that these vertically-transmitted microorganisms play an important role in plant fitness (Ferreira et al. 2008). This could point towards an evolved form of mutualism between the host plant and its microbial communities (Ewald 1987). It might well be that seed bacteria have exceptional abilities necessary for their survival inside the seed environment, and therefore only selected taxa are suitable to persist within plants over several generations. Indeed, microbiota typically found in seeds consist of only a limited number of bacterial species compared to other plant compartments such as the rhizosphere (Truyens et al. 2015). Several studies report the occurrence of a variety of bacterial seed endophytes belonging to Proteobacteria, mostly represented by the Gamma- and Alphaproteobacteria in seeds or caryopses of different plants (Barret et al. 2015; Klaedtke et al. 2016; Mitter et al. 2017). Members of Actinobacteria and Firmicutes have been also found within seeds, while other phyla like Bacteroidetes were reported, albeit less represented (Barret et al. 2015; Klaedtke et al. 2016; Mitter et al. 2017). Common bacterial genera isolated from inner seed/grain tissues of several plant species include Acinetobacter, Bacillus, Enterobacter, Micrococcus, Paenibacillus, Pantoea, Pseudomonas, Staphylococcus, and Stenotrophomonas (reviewed in Truyens et al. 2015). Bacterial seed endophytes may derive from a plethora of sources. Parts of the microbiota in plant seeds are considered to originate from the soil environment (Edwards et al. 2015), since taxa detected within seed tissues (that have not been in direct contact with the soil) show high similarity to common soil strains (Truyens et al. 2015). Additionally, it has been shown that the

terroir, which could be defined as a combination of environmental factors (e.g. soil type, climate) and human practices (Prévost and Lallemand 2010), is the key component shaping the composition of seed microbial assemblages (Klaedtke et al. 2016). Soil-associated microorganisms are attracted by exudates from the roots and may colonize the rhizosphere (Philippot et al. 2013). Passive or active mechanisms enable the entry of some bacterial endophytes into plant roots, depending on the colonizing strain (Compant et al. 2010; MercadoBlanco and Lugtenberg 2014). Once inside the root endosphere, vertical translocation of bacteria to aboveground plant compartments may take place via xylem vascular system (assisted by their flagella), by the transpiration streams, or throughout the mobilization along intercellular spaces (James et al. 2002; Compant et al. 2005). In this manner, bacterial migration towards the reproductive organs of angiosperms may occur, which has been demonstrated in the inner tissues of flowers (epidermis and ovary), fruit pulp and seeds of grapevine (Compant et al. 2011) and several other plant species, as reviewed by Hardoim et al. (2015). However, seed endophytes are not solely soil-derived. Alternative doorways for bacteria that might establish within inner seed tissues include entry points along external microenvironments of stems (caulosphere), flowers (anthosphere) as well as fruits (carposphere) or cones in the case of gymnosperms (Compant et al. 2010; Hardoim et al. 2015). Once established within the inner tissues of the maternal plant, translocation to the seed tissues may take place via vascular connections such as the funiculus and chalaza, or by the micropyle as reviewed by Truyens et al. (2015). Furthermore, microbial associations with gametes, like pollen grains, have also been reported in pine and pumpkin (Madmony et al. 2005; Fürnkranz et al. 2012; Ambika Manirajan et al. 2016), and may result in the colonization of the embryo and endosperm following pollination of the ovule (Agarwal and Sinclair 1996; Madmony et al. 2005). Colonization of meristematic tissues of buds by bacteria has been also reported and may allow some bacteria to be carried into seeds during differentiation, providing another route of colonization (Pohjanen et al. 2014). It is, however, noteworthy that bacterial communities associated with reproductive and disseminative organs of insect-pollinated plants are dominated by taxa rarely observed in other plant tissues (Shade et al. 2013; Ushio et al. 2015). This supports the notion that endophytic communities within reproductive and disseminative organs of allogamous

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plants could also derive from sources other than nearby colonized organs, and may include bacteria from airparticles or feeding insects (Hardoim et al. 2015; Lòpez-Fernàndez et al. 2017). In 1996, Maude described routes of colonization of seed-borne pathogens in angiosperms such as penetration through the ovary wall, systemic infection via the vascular system, penetration into the ovule via pollen germination inside the stigma (Maude 1996). Compant et al. (2011) discussed the presence of bacteria within seeds inside berries of grapevine and suggested that seed colonizing bacteria might originate either from the anthosphere or the soil and systemically spread in the plant and reach the ovule before seed development. Barret et al. (2016) discussed further the colonization and compartmentalization routes of various seedassociated plant pathogens and stated that when a pathogenic bacterium enters through the vascular system or floral pathways, it further colonizes all tissues within the seeds. However, colonization of mature seeds through contact with microorganisms present within the fruits or soils is usually restricted to the seed coat (Barret et al. 2016) if the seed has not yet germinated. Recently, Glassner et al. (2017) studied bacteria colonizing tissues of melon seeds and found taxa-specific colonization patterns. Alpha-, Beta-, Gammaproteobacteria, Firmicutes, and Actinobacteria were found in the outer or inner seed coat, and some were also present within the embryo including cotyledons and hypocotyl-root axis. Similarly, Alibrandi et al. (2017) demonstrated the presence of Alphaproteobacteria and Firmicutes in the inner seed coat and detected Firmicutes close to the vascular tissues of seeds of Anadenanthera colubrina. Examples of bacterial niches within seed tissues are shown in Figs. 1 and 2, and depict wheat kernels and tomato seeds showing colonization of surfaces, endosperm, and embryo by bacterial inhabitants. Differential amounts of bacteria have been observed not only among seeds of distinct plant species but also within seeds of the same genotypes (Rosenblueth et al. 2012; Klaedtke et al. 2016). Robinson et al. (2016) reported the quasiabsence of bacteria within embryos of wheat grain cv. Hereward, while they observed bacterial presence in other seed tissues such as the seed coat and endosperm, albeit with relatively low densities. This inter- and intraspecies seed-to-seed variability may depend on a wide range of factors like the conditions in which the parent plant was grown, the inter-individual heterogeneity of seed traits (such as seed size), and colonization events

throughout seed formation, development, and maturation (discussed by Klaedtke et al. 2016). Recently, Mitter et al. (2017) demonstrated transmission of bacteria in angiosperms from flowers to seeds by inoculating the flowers of different plant species with the beneficial endophyte, P. phytofirmans PsJN, which resulted in the establishment of this bacterium within the inner tissues of the successive seeds and seedlings. The same study showed that incorporating exogenous microorganisms into seeds may result in the alteration of the intrinsic seed microbial communities. However, the response of the seed microbiota to exogenous microorganisms may differ in response to the nature of the invader (Rezki et al. 2016). Mitter et al. (2017) further discussed that the success in the transmission of bacteria from flowers to seeds may depend on the flower age, condition of the stigma as well as the advance of the fertilization process. Likewise, the temporal window, during which bacteria colonize plant tissues that might later differentiate into seeds, is relatively narrow. One can further infer that seed colonization by bacteria (and consequently the composition of seed bacterial communities) may be subjected to random events and depend on a complex interplay of many different environmental factors. In summary, all the previous mentioned evidences delineated seeds/and grains as host organs of specific bacteria deriving from the anthosphere and carposphere in angiosperms, or pollen and ovules in cones of gymnosperms, as well as from inner tissues of plants following colonization from the soil, but also the possibility of colonization of meristem tissues prior to differentiation into reproductive and disseminative organs. The bacterial microbiota of plant seeds is a fascinating research field because the seed, like no other plant organ, may offer the possibility to design a plant with an optimized microbiome. However, the current knowledge on the ecology of seed endophytes is limited and yet does not allow rational design of seed microbiota. Current research in this field needs to be directed towards a better understanding of (i) the factors driving bacterial seed colonization, and (ii) the taxa that are able to colonize, survive and proliferate in seeds. Further challenges include the elucidation of optimal time frames and environmental conditions required for successful incorporation of microbes within distinct niches among the different seed tissues. Additional efforts in investigating the interactions between the incorporated microbes and the seed endogenous assemblages may be

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Fig. 1 Microphotographs of kernels of Triticum aestivum L. cv. Tschermakianum MANSF showing the presence of green fluorescent bacteria on surfaces of kernels, inside endosperm, and embryo

after Syto9® staining and confocal laser scanning microscopy. Similar finding was obtained for inner tissues using surface sterilized kernels or not

necessary. Do these seed microbiota interfere with each other or do they facilitate the colonization of certain strains? Some bacterial endophytes, for instance, occur in a viable but not cultivable state and might be reactivated by an exogenous microorganism or by specific environmental factors (Podolich et al. 2015). Many members of the seed microbiota have been detected

using molecular approaches but also lack culturability. Efforts in obtaining bacterial isolates from seeds could shed light on the roles of these communities in the growth and health of the plant at different developmental stages. Genomic analyses of seed bacterial endophytes may also provide valuable information that might help clarify the above-mentioned processes, and

Fig. 2 Microphotographs of Solanum lycopersicum L. cv. Moneymaker seeds showing the presence of green fluorescent bacteria on surfaces of the seed, inside the endosperm, leaf, and root

embryo after Syto9® staining and confocal laser scanning microscopy. Similar finding was obtained for inner tissues using surface sterilized seeds or not

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therefore represent an important aspect that requires investigation. We stand before a pivotal point in the elucidation of seed microbiota and their characteristic colonization behaviors, thus plenty of research regarding these peculiar microbial assemblages is highly encouraged, since it may lead to future applications that modulate plant health and development employing naturally occurring colonization routes and traits of microorganisms from the very beginning of the plant’s life. Acknowledgements This work is supported by a grant from the Austrian Science Foundation (FWF P 26203) to AS. Compliance with ethical standards Conflict of interest Stéphane Compant and Birgit Mitter are Section Editors in Plant and Soil and Stéphane Compant, Matthieu Barret, and Birgit Mitter are Guest Editors of the special issue. This does not, however, interfere with the reviewing process.

References Agarwal VK, Sinclair JB (1996) Principles of seed pathology. Lewis Publication, Boca Raton Alibrandi P, Cardinale M, Rahman MM, Strati F, Ciná P, de Viana ML, Giamminola EM, Gallo G, Schnell S, de Filippo C, Ciaccio M, Puglia AM (2017) The seed endosphere of Anadenanthera colubrina is inhabited by a complex microbiota, including Methylobacterium spp. and Staphylococcus spp. with potential plant-growth promoting activities. Plant Soil 1–19. doi:10.1007/s11104-017-3182-4 Ambika Manirajan B, Ratering S, Rusch V, Schwiertz A, GeisslerPlaum R, Cardinale M, Schnell S (2016) Bacterial microbiota associated with flower pollen is influenced by pollination type, and shows a high degree of diversity and species-specificity. Environ Microbiol 18: 5161–5174. doi:10.1111/1462-2920.13524 Barret M, Briand M, Bonneau S, Préveaux A, Valière S, Bouchez O, Hunault G, Simoneau P, Jacques M-A (2015) Emergence shapes the structure of the seed microbiota. Appl Environ Microbiol 81:1257–1266 Barret M, Guimbaud J-F, Darrasse A, Jacques M-A (2016) Plant microbiota affects seed transmission of phytopathogenic microorganisms. Mol Plant Pathol 17:791–795 Compant S, Reiter B, Sessitsch A, Nowak J, Clément C, Ait Barka E (2005) Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN. Appl Environ Microbiol 71:1685–1693. doi:10.1128/aem.71.4.1685-1693.2005 Compant S, Clément C, Sessitsch A (2010) Plant growthpromoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol Biochem 42: 669–678. doi:10.1016/j.soilbio.2009.11.024

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. Microb Ecol 62:188–197. doi:10.1007/s00248-011-9883-y Edwards J, Johnson C, Santos-Medellín C, Lurie E, Podishetty NK, Bhatnagar S, Eisen JA, Sundaresan V (2015) Structure, variation, and assembly of the root-associated microbiomes of rice. Proc Natl Acad Sci U S A 112: 911–920. doi:10.1073/pnas.1414592112 Ewald PW (1987) Transmission modes and evolution of the parasitism-mutualism continuum. Ann N Y Acad Sci 503: 295–306 Ferreira A, Quecine MC, Lacava PT, Oda S, Azevedo JL, Araújo WL (2008) Diversity of endophytic bacteria from eucalyptus species seeds and colonization of seedlings by Pantoea agglomerans. FEMS Microbiol Lett 287:8–14. doi:10.1111/j.1574-6968.2008.01258.x Fürnkranz M, Lukesch B, Müller H, Huss H, Grube M, Berg G (2012) Microbial diversity inside pumpkins: microhabitatspecific communities display a high antagonistic potential against phytopathogens. Microb Ecol 63:418–428. doi:10.1007/s00248-011-9942-4 Glassner H, Zchori-Fein E, Yaron S, Sessitsch A, Sauer U, Compant S (2017) Bacterial niches inside seeds of Cucumis melo L. Plant Soil:1–13. doi:10.1007/s11104-017-3175-3 Hardoim PR, Hardoim CCP, van Overbeek LS, van Elsas JD (2012) Dynamics of seed-borne rice endophytes on early plant growth stages. PLoS One 7:e30438. doi:10.1371/journal.pone.0030438 Hardoim PR, van Overbeek LS, Berg G, Pirttilä AM, Compant S, Campisano A, Döring M, Sessitsch A (2015) The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 79:293–320. doi:10.1128/MMBR.00050-14 James EK, Gyaneshwar P, Mathan N, Barraquio WL, Reddy PM, Iannetta PP, Olivares FL, Ladha JK (2002) Infection and colonization of rice seedlings by the plant growth-promoting bacterium Herbaspirillum seropedicae Z67. Mol Plant-Microbe Interact 15: 894–906. doi:10.1094/MPMI.2002.15.9.894 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. doi:10.1371/journal.pone.0020396 Klaedtke S, Jacques M-A, Raggi L, Préveaux A, Bonneau S, Negri V, Chable V, Barret M (2016) Terroir is a key driver of seedassociated microbial assemblages. Environ Microbiol 18: 1792–1804. doi:10.1111/1462-2920.12977 Lòpez-Fernàndez S, Mazzoni V, Pedrazzoli F, Pertot I, Campisano A (2017) A phloem-feeding insect transfers bacterial endophytic communities between grapevine plants. Front Microbiol 15; 8:834. 10.3389/fmicb.2017.00834 Madmony A, Chernin L, Pleban S, Peleg E, Riov E (2005) Enterobacter cloacae, an obligatory endophyte of pollen grains of Mediterranean pines. Folia Microbiol (Praha) 50: 209–216 Maude RB (1996) Seedborne diseases and their control: principles and practice. CAB International, Wallingford

Plant Soil Mercado-Blanco J, Lugtenberg BJJ (2014) Biotechnological applications of bacterial endophytes. Curr Biotechnol 3:60–75 Miller IM (1990) Bacterial leaf nodule symbiosis. Adv Bot Res Adv Plant Pathol 17:163–234 Mitter B, Pfaffenbichler N, Flavell R, Compant S, Antonielli L, Petric A, Berninger T, Muhammad N, Sheibani-Tezerji R, Von Maltzahn G, Sessitsch A (2017) A new approach to modify plant microbiomes and traits by introducing beneficial bacteria at flowering into progeny seeds. Front Microbiol. doi:10.3389/fmicb.2017.00011 Philippot L, Raaijmakers JM, Lemanceau P, van der Putten WH (2013) Going back to the roots: the microbial ecology of the rhizosphere. Nat Rev Microbiol 11:789–799 Podolich O, Ardanov P, Zaets I, Pirttilä A, Kozyrovska N (2015) Reviving of the endophytic bacterial community as a putative mechanism of plant resistance. Plant Soil 388(1–2):367–377 Pohjanen J, Koskimäki JJ, Pirttilä AM (2014) Interactions of meristem-associated endophytic bacteria. In: Verma VC, Gange AC (eds) Advances in endophytic research. New Delhi, Springer India, pp 103–113 Prévost P, Lallemand P (2010) L’approche terroir : pour une démarche de recherche-formation-action http://hal.archives-ouvertes. fr/docs/00/51/25/28/PDF/Prevost_approche-terroir.pdf Rezki S, Campion C, Iacomi-Vasilescu B, Preveaux A, Toualbia Y, Bonneau S, Briand M, Laurent E, Hunault G, Simoneau P, Jacques M-A, Barret M (2016) Differences in stability of seed-associated microbial assemblages in response to invasion by phytopathogenic microorganisms. PeerJ 4:e1923 Robinson RJ, Fraaije BA, Clark IM, Jackson RW, Hirsch PR, Mauchline TH (2016) Wheat seed embryo excision enables

the creation of axenic seedlings and Koch’s postulates testing of putative bacterial endophytes. Sci Rep 6:25581 Rosenblueth M, López-López A, Martínez J, Rogel MA, Toledo I, Martínez-Romero E (2012) Seed bacterial endophytes: common genera, seed-to-seed variability and their possible role in plants. Acta Hort (ISHS) 938:39–48 Shade A, McManus PS, Handelsman J (2013) Unexpected diversity during community succession in the apple flower microbiome. MBio 4:e00602–e00612. doi:10.1128/mBio.00602-12 Smith EF (1911) Bacteria in relation to plant diseases. Carnegie institution of Washington, Washington, D.C. Truyens S, Weyens N, Cuypers A, Vangronsveld J (2015) Bacterial seed endophytes: genera, vertical transmission and interaction with plants. Environ Microbiol Rep 7:40–50. doi:10.1111/1758-2229.12181 Ushio M, Yamasaki E, Takasu H, Nagano A, Fujinaga S, Honjo MN, Ikemoto M, Sakai S, Kudoh H (2015) Microbial communities on flower surfaces act as signatures of pollinator visitation. Sci Rep 5:8695. doi:10.1038/srep08695 Van Oevelen S, De Wachter R, Vandamme P, Robbrecht E, Prinsen E (2002) Identification of the bacterial endosymbionts in leaf galls of Psychotria (Rubiaceae, angiosperms) and proposal of ‘Candidatus Burkholderia kirkii’ sp. nov. Int J Syst Evol Microbiol 52:2023–2027 Van Oevelen S, De Wachter R, Vandamme P, Robbrecht E, Prinsen E (2004) ‘Candidatus Burkholderia calva’ and ‘Candidatus Burkholderia nigropunctata’ as leaf gall endosymbionts of African Psychotria. Int J Syst Evol Microbiol 54:2237–2239 von Faber FC (1912) Das erbliche Zusammenleben von Bakterien und tropischen Pflanzen. Jahrb Wiss Bot 54:285–375