Received: 16 July 2019 | Revised: 25 September 2019 | Accepted: 27 September 2019 DOI: 10.1002/mbo3.954 ORIGINAL ARTICLE The ecological role of bacterial seed endophytes associated with wild cabbage in the United Kingdom Olaf Tyc1,2 | Rocky Putra1,3,4 | Rieta Gols3 | Jeffrey A. Harvey5,6 | Paolina Garbeva1 1Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO- Abstract KNAW), Wageningen, The Netherlands Endophytic bacteria are known for their ability in promoting plant growth and de- 2 Department of Internal Medicine I, Goethe fense against biotic and abiotic stress. However, very little is known about the micro- University, University Hospital Frankfurt, Frankfurt, Germany bial endophytes living in the spermosphere. Here, we isolated bacteria from the seeds 3Laboratory of Entomology, Wageningen of five different populations of wild cabbage (Brassica oleracea L) that grow within University, Wageningen, The Netherlands 15 km of each other along the Dorset coast in the UK. The seeds of each plant popu- 4Hawkesbury Institute for the Environment, Western Sydney University, Penrith, lation contained a unique microbiome. Sequencing of the 16S rRNA genes revealed Australia that these bacteria belong to three different phyla (Actinobacteria, Firmicutes, and 5Department of Terrestrial Ecology, Proteobacteria). Isolated endophytic bacteria were grown in monocultures or mix- Netherlands Institute of Ecology, Wageningen, The Netherlands tures and the effects of bacterial volatile organic compounds (VOCs) on the growth 6 Department of Ecological Sciences, Section and development on B. oleracea and on resistance against a insect herbivore was Animal Ecology, VU University Amsterdam, Amsterdam, The Netherlands evaluated. Our results reveal that the VOCs emitted by the endophytic bacteria had a profound effect on plant development but only a minor effect on resistance against Correspondence Olaf Tyc, Department of Microbial Ecology, an herbivore of B. oleracea. Plants exposed to bacterial VOCs showed faster seed ger- Netherlands Institute of Ecology (NIOO- mination and seedling development. Furthermore, seed endophytic bacteria exhib- KNAW), PO BOX 50, 6,700 AB Wageningen, The Netherlands. ited activity via volatiles against the plant pathogen F. culmorum. Hence, our results Department of Internal Medicine I, Goethe illustrate the ecological importance of the bacterial seed microbiome for host plant University, University Hospital Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, health and development. Germany. Email: [email protected] KEYWORDS bacteria, endophytes, fungal pathogens, insect herbivory, plant growth, plant resistance, Funding information Nederlandse Organisatie voor plant-insect interactions, seed germination Wetenschappelijk Onderzoek, Grant/Award Number: 864.11.015 1 | INTRODUCTION & Martinez-Romero, 2006). Endophytic microorganisms live in an intimate relationship with their host throughout many generations Plants are involved in intimate interactions with microbes through- (Johnston-Monje & Raizada, 2011). Many endophytic bacteria are out their entire life cycle, and these interactions are essential for known for their growth-promoting effect on plants and for priming the growth and health of the plants. Endophytes are nonpatho- plant immunity by triggering induced systemic resistance (ISR) and/ genic microorganisms that inhabit plants without causing them any or induced systemic tolerance (IST) (Hardoim, Overbeek, & Elsas harm (Hardoim, Hardoim, Overbeek, & Elsas, 2012; Rosenblueth JDv., 2008; Nass et al., 2005; Ryu et al., 2004, 2003). Several studies This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. MicrobiologyOpen. 2020;9:e954. www.MicrobiologyOpen.com | 1 of 19 https://doi.org/10.1002/mbo3.954 2 of 19 | TYC ET AL. have shown that endophytes can enhance antagonistic activities of Here, we aimed to investigate the potential role of volatiles plant pathogens and aid plants against biotic and abiotic stresses produced by seed endophytic bacteria associated with wild cab- (Berg et al., 2005; Cosme et al., 2016; Egamberdieva, Davranov, bage (Brassica oleracea L.) on plant growth, development and resis- Wirth, Hashem, & Abd_Allah EF., 2017; Grover, Ali, Sandhya, Rasul, tance against a leaf chewing insect herbivore and two pathogenic & Venkateswarlu, 2011). Seed endophytes are known to be verti- fungi. These wild cabbage populations are considered to be the cally transmitted from mother plants to their offspring (Hardoim, ancestors of current cultivated cabbage. Seeds originated from Overbeek and Elsas, 2008; Frank, Saldierna Guzmán and Shay, 2017; five populations growing along the rugged coastline of Dorset, Nelson, 2018), suggesting that the role of seed endophytes is highly United Kingdom (Gols, Dam, Raaijmakers, Dicke, & Harvey, crucial, especially at the early stage of host plant development (Berg 2009; Van Geem, Harvey, Cortesero, Raaijmakers, & Gols, 2015; & Raaijmakers, 2018; Nelson, 2018; Truyens, Weyens, Cuypers, & Wichmann, Alexander, Hails, & Bullock, 2008). Previous work has Vangronsveld, 2015). However, little is known so far about the eco- shown that there is considerable population-related variation in logical role of the seed endophytes and seed microbiome. the expression of primary and secondary metabolites (glucosino- Essential for plant–microbe interactions and communication are lates) in British populations of wild cabbage. These differences secondary (or specialized) metabolites produced by either partners. have an effect on the behavior and development of several spe- Both plants and microorganisms produce a wide variety of second- cies of insect herbivores and their natural enemies associated with ary metabolites including volatile and nonvolatile compounds. Only these plants both in the laboratory and in the field (Gols, Bullock, in the past few decades, the functional role of microbial volatiles Dicke, Bukovinszky, & Harvey, 2011; Gols et al., 2008; Harvey, has been increasingly acknowledged and investigated (Sharifi & Ryu, Dam, Raaijmakers, Bullock, & Gols, 2011; Moyes, Collin, Britton, 2018). Plant-associated microorganisms produce an vast array of & Raybould, 2000; Newton, Bullock, & Hodgson, 2009; Van Geem volatiles ranging from inorganic compounds, such as CO2, NH3, and et al., 2015). However, this previous research ignored the possibly HCN to a plethora of organic compounds, such as terpenes, ketones, important role played by the plant microbiome on plant traits that alcohols, alkenes, alkanes, esters, and sulfur-derived compounds affect growth, fitness, and defense. We hypothesize that seeds of (Kanchiswamy, Malnoy, & Maffei, 2015; Schulz & Dickschat, 2007). wild cabbage contain cultivable endophytic bacteria whose vola- Volatile organic compounds (VOCs) are compounds with a small mo- tiles are beneficial for the host plant. Here, we aim to isolate en- lecular weight (<300 da). They can easily evaporate and travel through dophytic bacteria from five different populations of wild cabbage air and water-filled pores in the soil (Penuelas et al., 2014; Schulz & plant populations. We hypothesize that the five different plant Dickschat, 2007). So far, the most well studied VOCs emitted by soil populations harbor different endophytic bacterial strains, each microorganisms are terpenes, nitrogen-based compounds like indole producing its specific volatile blend, which in turn differentially and sulfur-containing compounds like dimethyl disulfide (Tyc, Song, affect their interaction with the host plant. Dickschat, Vos, & Garbeva, 2017). Soil microorganisms can employ volatiles as info chemicals, growth stimulants, growth inhibitors, and inhibitors of quorum sensing (Chernin et al., 2011; Effmert, Kalderas, 2 | MATERIALS AND METHODS Warnke, & Piechulla, 2012; Kai et al., 2009; Kim, Lee, & Ryu, 2013). Moreover, interspecific interactions of phylogenetically different 2.1 | Seeds and extraction of endophytic bacteria bacteria can also alter the volatile blend composition, affecting the activity of volatiles (Garbeva, Hordijk, Gerards, & Boer, 2014; Tyc Seeds of five different populations of wild cabbage Brassica olera- et al., 2015). The effects of the emitted microbial VOCs on the host cea collected from the Dorset coast in the UK were used in this plants and their antagonists can vary from negative, positive to study: A: Durdle Door (DD; 50˚62’N, 2˚27’W), B: Kimmeridge neutral (van Dam, Weinhold, & Garbeva, 2016). For instance, plant- (KIM; 50˚35’N, 2˚03’W), C: Old Harry (OH; 50˚38’N, 1˚55’W), D: growth promoting effects were reported for volatiles emitted by bac- St. Aldhelms Head (SAH; 50˚69’N, 2˚05’W), and E: Winspit (WIN; teria (Park, Dutta, Ann, Raaijmakers, & Park, 2015; Ryu et al., 2003) 50˚34’N, 2˚02’W) (Van Geem et al., 2015) (Figure 1a; Figure A1). and fungi (Cordovez et al., 2017). In addition, volatiles from an endo- Seeds were surface-sterilized by a modified protocol by Araujo et phyte of maize (Zea mays), Enterobacter aerogenes have been shown al. (2002). To this end, seeds (1 g) of each plant population were to alter the host plant's resistance to a fungal pathogen and an insect subsequently incubated for 3 min in 2% NaOCl, 3 min in 80% etha- pest (D'Alessandro et al., 2014), suggesting that volatiles also exhibit nol, and rinsed five times