Project 1: Going back to the roots

Deciphering seed & root microbiomes of crop species and their wild relatives for beneficial consortia

•  Project leader: Prof. Dr. Jos M. Raaijmakers, NIOO-KNAW

I. SCIENTIFIC DESCRIPTION

Introduction

The spermosphere and rhizosphere are the narrow zones (2-10mm) surrounding and influenced by plant seeds and roots, respectively (Nelson, 2004; Mendes et al. 2012; Philippot et al. 2013). Associations of plants with beneficial and/or pathogenic microorganisms, in most cases, start in the spermosphere yet little is known about the diversity, succession, and activities of indigenous microbial communities on/in seeds and how this short-lived, dynamic developmental stage affects microbiome assembly in the rhizosphere when plants grow older. This limited knowledge is one of the main reasons for the frequent failure of many promising microbial inoculants studied in the past, resulting in poor establishment, colonization, survival and performance under real agricultural & horticultural conditions. Furthermore, there has been too much emphasis to date on one-microbe-at-a-time applications, whereas most ecosystem functions are generally driven by the activity of microbial consortia. Recent studies at NIOO have indeed shown that several microorganisms that do not have a specific activity alone, exhibit antimicrobial activity when they are part of a consortium (Garbeva et al. 2011). Hence, a better fundamental understanding of spermosphere and rhizosphere microbiology and chemistry is urgently needed to improve our selection of beneficial microbial consortia that are highly compatible with seeds and roots of a given crop/plant species or cultivar. Furthermore, comparative microbial community analyses of the spermosphere and rhizosphere of crop species and their wild relatives will provide insight into core and accessory microbiomes of a given crop/plant species. This knowledge can subsequently be exploited via plant breeding and/or by seed technology such as designer seed coatings to manipulate spermosphere and rhizosphere habitats towards selection and activation of specific beneficial microbial consortia.

Major aims of this project

1. Decipher the structural and functional diversity as well as the temporal dynamics of microbial communities in the spermosphere and rhizosphere of crop species and their wild relatives grown in agricultural and/or native habitats
2. Characterize specific microbial genera and consortia for their beneficial effects on seed germination, seedling vigour, plant growth and root development.
3. Identify constituents in seed and root exudates that trigger the growth and activity of specific beneficial microbial genera (in collaboration with project 4).
4. Generate knowledge on microbial traits/genes for tailor-made microbe-crop combinations for better plant growth and enhanced tolerance to (a)biotic stress (in collaboration with projects 2 & 3).

Research plan

Workpackage 1.1  Structural and functional diversity of seed and root microbiomes:

• Seed germination generally progresses through three distinct phases, going from imbibition to extension and protrusion of the radicle through the seed coat (Nelson, 2004). The period of these phases is determined by intrinsic seed properties and environmental factors. Phase I (imbibition) and phase III (radicle protrusion) are accompanied by bursts of exudates that may consist of diverse sugars, amino acids, organic acids, phenolic compounds and volatile organic compounds (VOCs) (Nelson, 2004). To date, relatively little is known about the exudate composition and VOCs profiles of seeds of different crops and which microbial communities and functions are attracted and activated, especially during the first 24 h of seed germination.
• To decipher the structural and functional diversity as well as the succession of microbial communities in the spermosphere and rhizosphere of crop species, large-scale metagenomic/transcriptomic analyses will be conducted in this project. Seeds of several crop species (tomato, lettuce, soya, cabbage, onion, maize) will be sown in small batches of agricultural or horticultural relevant soils and incubated under controlled conditions. For several of these crops, wild relatives/ancestors will be included; the choice of the crop ancestors will be made based on the available expertise and seed repositories of the participating companies. For two different developmental stages of germination (Phase I & III) and one stage of seedling growth (first true leaves), microbes and their DNA and RNA will be isolated from the surface (spermoplane, rhizoplane) of germinating seeds and plant roots using standard extraction and isolation protocols. DNA and cDNA libraries will be prepared for 16S-amplicon and shotgun sequencing. Sequence analyses and statistics will be carried out using diverse tools, among others, QIIME, PhyloseqR, STAMP, MG-RAST, MOTHUR and specific in-house developed scripts and Galaxy-based pipelines at NIOO.
• Based on these metagenomic and transcriptomic analyses, specific bacterial genera and consortia that are abundantly present and active across the three developmental stages of germination and seedling growth of crops and their wild relatives/ancestors, will be subjected for targeted isolation on (semi)selective media and tested alone and in consortia for their beneficial effects on seed germination, plant growth and tolerance to (a)biotic stress (drought & pathogens) with the other partners (projects 2 & 3).
• For the crop species and wild relatives tested, exudates will be extracted/collected from seeds (Phase I, III) germinating in y-irradiated soils and analysed chemically for sugars, amino acids, fatty acids, secondary metabolites and VOCs. Both at NIOO and at IBL (LU), the required chemical analytical infrastructure (LC-MS/MS, GC-QTOF, IRMS, NMR) and expertise are available to resolve the identity of various seed exudate components. Fractionated exudate components will be tested for their effects on growth and chemotaxis of the isolated bacteria with beneficial effects on seed germination and seedling growth. Effects of seed exudate fractions and specific components on the induction of specific genes in the isolated bacterial consortia will be tested by transcriptomics and obtained results will be compared to the initial metatranscriptome data described above. The role of selected, highly expressed bacterial genes in seed colonization and chemotaxis will be investigated via site-directed mutagenesis or heterologous expression (depending on the ability/efficiency to transform the bacterial genera). Finally, Mass Spectrometry Imaging (Watrous et al. 2011) will be conducted to determine if the identified activities and corresponding exudates and bacterial metabolites are produced in situ.
• Collectively, the results will provide fundamental insight into the genetic, functional and chemical diversity of microorganisms and exudates of seeds of economically important crop species and their wild relatives.

Workpackage 1.2 Endophytic microbiomes of crop species & wild relatives in their native habitats:

• Here we will test the hypothesis that wild relatives of crop species grown in their native soils support a more compatible and stable beneficial endophytic microbiome as compared to modern cultivars and agricultural soils. We will conduct an in-depth analysis of the endophytic bacterial community of seeds and roots of common bean (Phaseolus vulgaris) grown in their native and agricultural habitats in Colombia. Common bean is one of the most important legume food crops worldwide and is pivotal to the subsistence economy in Latin America and Africa (Akibode and Maredia, 2011; Broughton et al., 2003; CGIAR, 2013). Its origin and domestication process has been extensively studied and represents an intriguing example of multiple domestication events. The origin of the wild common bean was clarified recently by nuclear and chloroplast DNA analyses, with central Mexico as the cradle of its diversity (Bitocchi et al., 2012; Desiderio et al., 2013). Subsequently, the wild common bean ancestor spread throughout Central and South America, reaching its current distribution from Mexico to Argentina. This resulted in two genetic pools (Mesoamerican, Andean) that come together in Colombia.
• Close collaboration has been initiated in 2013 with Prof. Camillo Ramirez of the University of Antioquia (Medellin, Colombia) and with Dr. Daniel DeBouck of the International Centre of Tropical Agriculture (CIAT, Palmira, Colombia) for their expertise in bean cultivation and phenology, their large and well documented collection of common bean accessions and their knowledge of agricultural and native habitats. Seeds of 9 bean accessions (3 wild relatives, 3 landraces, 3 modern cultivars) will be sown in soil from the Antioquia mountain area (native habitat) and from Colombian agricultural soil commonly used for bean cultivation. These soils already have been collected and characterized for physic-chemical properties. Screenhouse assays will be conducted at the University of Antioquia. Germinating seeds (Phase I, III) and plant roots (true leaf stage and flowering stage) will be harvested and surface sterilized to remove epiphytic microorganisms. In-house protocols for surface sterilization are available as well as a Nycodenz-based protocol to collect and separate endophytic microbial cells from plant cells of internal seed and root tissues. The diversity, succession and functional potential of the seed and root endophytic microbiome will be characterized by the metagenomic and microbiological methodologies described in WP 1.1. Effects on seed germination, seedling vigour, plant growth and development, and tolerance to (a)biotic stress (drought & pathogens) will be conducted with the other partners (projects 2 & 3).
• Bacterial strains from consortia that are highly beneficial for plant growth and health will be subjected to genome sequencing and mutagenesis (depending on the transformation efficiency) to identify genes and traits involved in seed germination, seedling growth and root development.

II. UTILISATION PLAN