Volatile-Mediated Interactions in the Rhizosphere

Volatile-mediated interactions in the rhizosphere Viviane Cordovez da Cunha Thesis committee Promotors Prof. Dr F.P.M. Govers Personal Chair at the Laboratory of Phytopathology Wageningen University Prof. Dr J. M. Raaijmakers Professor of Microbial Ecology Leiden University Head of the Department of Microbial Ecology Netherlands Institute of Ecology, Wageningen Co-promotor Dr V. J. Carrion Researcher at the Department of Microbial Ecology Netherlands Institute of Ecology, Wageningen Other members Prof. Dr J.J.A. van Loon, Wageningen University Dr K. Vrieling, Leiden University Prof. Dr J.S. Dickschat, University of Bonn, Germany Prof. Dr L. Eberl, University of Zürich, Switzerland This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences. Volatile-mediated interactions in the rhizosphere Viviane Cordovez da Cunha Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 4th November 2016 at 4 p.m. in the Aula. Viviane Cordovez da Cunha Volatile-mediated interactions in the rhizosphere, 220 pages. PhD thesis, Wageningen University, Wageningen, NL ( 2016) With references, with summaries in English, Dutch and Portuguese ISBN 978-94-6257-901-9 DOI: http://dx.doi.org/10.18174/388360 Table of Contents Chapter 1 General introduction and thesis outline 7 Chapter 2 Volatile affairs in microbial interactions 21 Chapter 3 Diversity and functions of volatile organic compounds 33 produced by Streptomyces from a disease-suppressive soil Chapter 4 Genomic and functional analyses of rhizospheric and 61 endophytic Microbacterium species Chapter 5 Exploring volatile organic compounds of Microbacterium 95 for plant growth promotion and biocontrol Chapter 6 Volatiles from a soil-borne pathogenic fungus modulate 111 the trade-off between plant growth and insect resistance Chapter 7 General discussion 155 References 167 Summary 187 Samenvatting 193 Resumo 199 Acknowledgments 205 About the author 211 Publications 211 Education statement 215 Chapter 1 General introduction and thesis outline CHAPTER ONE | 9 Volatile organic compounds (VOCs) are carbon containing molecules of low molecular weight and high vapor pressure that can easily evaporate and diffuse through soil and atmosphere. Due to these physical-chemical properties, they can facilitate interactions between spatially separated organisms. Plants produce a wide variety of VOCs that mediate multiple interactions with their environment, ranging from the attraction of pollinators and seed dispersers (Dudareva et al., 2004; Knudsen et al., 2006) to plant defense and plant-plant communication (Paré & Tumlinson, 1999; Mumm & Dicke, 2010). Although VOCs produced by microorganisms have received attention only in the past decade, these compounds have been long described in literature. Zoller and Clark (1921) reported for the first time the production of VOCs by bacteria of the dysentery group. Five years later, Russian researchers reported that the ‘soil air’ should be recognized as an organic constituent, equally important as the soil aqueous and solid phases (Curl & Truelove, 1986). Several studies from the late 1960s and early 1970s described the presence of ‘volatile inhibitors’ in soil. For example, microbial volatile organic and inorganic compounds were reported as major factors in soil fungistasis, the widespread soil characteristic that prevents viable fungal spores from germinating (Dobbs & Hinson, 1953). Fungistastic effects of the soil were relieved by partial sterilization and addition of antibiotics (Epstein & Lockwood, 1984), indicating that these VOC-mediated effects were of microbial origin. Hora and Baker (1972) demonstrated the production of a ‘volatile inhibitor’ of fungal spore germination in sterilized soils inoculated with soil actinomycetes and individual isolates of Trichoderma. Since then, several soil VOCs have been shown to reduce or inhibit spore germination and hyphal growth of fungi (Chuankun et al., 2004; van Agtmaal et al., 2015) and to influence plant growth and development (Vespermann et al., 2007; Bailly & Weisskopf, 2012). Diversity and biosynthesis of microbial volatiles Currently, more than 1000 bacterial and fungal VOCs have been described in literature (Effmert et al., 2012). Microbial VOCs typically include alkenes, alcohols, ketones, terpenes, benzenoids, pyrazines, acids, esters, and aldehydes (Piechulla & Degenhardt, 2014). Many of these compounds are produced by different soil and plant-associated bacteria and fungi (Fig. 1). Some of the microbial VOCs are common to several phylogenetic groups, while others seem to be unique for some species (Larsen & Frisvad, 1995; Schnürer et al., 1999; Muller et al., 2013). One of the most well-known microbial VOCs is geosmin, produced by most Streptomyces species, cyanobacteria and some fungi. Geosmin is a member of the terpene class of compounds and is responsible for the characteristic musty or earthy smell 10 | General introduction and thesis outline of moist soils (Gerber, 1968; Jiang et al., 2007). Although this compound has been identified already in 1891 and isolated in 1965, its ecological roles remain unknown to date (Berthelot & Andre, 1891; Gerber, 1968; Jiang et al., 2007). Microbial VOCs are produced via both primary and secondary metabolism (Korpi et al., 2009). The biosynthetic pathways involved in VOC production by microorganisms are carbon metabolism, fatty acid degradation, fermentation, amino acid catabolism, terpenoid biosynthesis, and sulfur metabolism (Peñuelas et al., 2014). Microbial production of aliphatic hydrocarbons is typically derived from fatty acids (Schulz & Dickschat, 2007). Decarboxylation of intermediates results in alkanes, alkenes or methyl ketones, whereas the reduction of the carboxyl group leads to the generation of aldehydes and alkanols (Peñuelas et al., 2014). Eight-carbon VOCs, such as 1-octen-3-ol and 3-octanone, are products of the oxidation and cleavage of the fatty acid linoleic acid, and classified as oxylipins (Combet et al., 2006). Acetoin (3-hydroxy-2-butanone) and its oxidized form 2,3- butanedione are derived from pyruvate fermentation under anaerobic conditions (Ryu et al., 2003; Audrain et al., 2015). Short-chain-branched alcohols, such as 3-methyl-1-butanol and 2-methyl-1- butanol, are produced by enzymatic conversion of branched chain amino acids via the Ehrlich pathway (Marilley & Casey, 2004). Aromatic hydrocarbons, such as 2-phenylethanol, are generated in bacteria and fungi via the shikimic acid pathway, with L-phenylalanine as a precursor (Hazelwood et al., 2008; Kim et al., 2014). The bacterial production of indole, another aromatic hydrocarbon, is regulated by several environmental factors such as the availability of extracellular tryptophan, cell population density, catabolite repression, temperature and pH (Lee & Lee, 2010). The indole biosynthetic pathway is well characterized in Escherichia coli, where tryptophanase encoded by tnaA converts tryptophan into indole, pyruvate and ammonia (Newton & Snell, 1965; Pittard, 1996). Aliphatic amino acids, such as glutamate, aspartate, alanine, valine, leucine, isoleucine and methionine, serve as precursors for bacterial VOCs. Decarboxylation of these amino acids followed by deamination results in their respective 2-keto acids. After decarboxylation and in the presence of 2-keto acid decarboxylase, they are then transformed into alcohols and/or into ethyl or methyl esters (Roze et al., 2010). Terpenes are the largest class of natural VOCs with over 50.000 known members (Conolly & Hill, 1991). They can mediate numerous antagonistic and beneficial interactions among (micro)organisms (Gershenzon & Dudareva, 2007; Dickschat et al., 2014). Fungi and bacteria produce a variety of terpenoids which are transformed and rearranged to a multitude of compounds (Kramer & Abraham, 2011; Cane & Ikeda, 2012; Song et al., 2015). Terpene synthases are the primary enzymes responsible for catalyzing the formation of CHAPTER ONE | 11 hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15) or diterpenes (C20) from the substrates dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP) farnesyl diphosphate (FPP) and geranylgeranly diphosphate (GGPP), respectively (Tholl, 2006; Dickschat et al., 2014). Several terpene cyclases and synthases have been identified by mining of bacterial genomes, including those involved in the biosynthesis of 2- methylisoborneol (2-MIB) and 2-methylenebornane (Komatsu et al., 2008; Chou et al., 2010; Moody et al., 2012; Yamada et al., 2015). Sulfur-containing VOCs range from relatively small compounds such as dimethyl sulfide, dimethyl disulphide and dimethyl trisulphide to more complex VOCs, such as 2- methyl-4,5-dihydrothiophene (Splivallo et al., 2011; Effmert et al., 2012). Two main biosynthetic pathways have been described for the production of sulfur-containing VOCs. The first pathway involves a one-step conversion of L-methionine to metanethiol by methionine lyases. The second one is initiated by L-methionine transamination to 4- methylthio- 2-oxobutyric acid, which is then converted to 3-(methylthio)propanal via decarboxylation or reduced to 4-methylthio-2-hydroxybutyric acid, with the ultimate formation of metanethiol (Bustos et al., 2011; Splivallo et al., 2011). Dimethyl sulfide is formed by the cleavage of dimethylsulfoniopropionate,

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