Project 3: Microbial support of plant growth under abiotic stress

Identifying microbes and mechanisms involved in drought tolerance of plants

 

I. SCIENTIFIC DESCRIPTION

 

Introduction

Microbial communities at seed- and root-soil interfaces (epiphytes) as well as inside the plant (endophytes) can increase plant fitness under diverse environmental growth conditions, including abiotic stresses like drought, heat and salinity (Yang et al., 2009; Gover et al., 2010). We argue that the contribution of the spermosphere and rhizosphere to such resistance is most prevalent in harsh environments. Deserts represent such ecological niche and may contain a diversity of yet unknown microbes that improve plant performance under abiotic stress and can be used on crops. For example, the endophytic fungus Piriformospora indica and soil bacterium Achromobacter piechaudii, both isolated from arid and saline environments, confer abiotic stress tolerance to a broad range of host plants (Mayak et al., 2004; Varma et al., 2013). In line with this, we expect that deserts harbor a more diverse range of microbial networks that help local vegetation to resist abiotic stress. However, only a limited number of studies have been conducted to characterize these microbial communities (e.g. Koberl et al., 2011; Marasco et al., 2012). The main objective of this project is to identify the core microbiomes that form the spermosphere and rhizosphere in desert habitats and how these microbial consortia contribute to abiotic stress tolerance of crop plants at different stages of their development. To do so, we will focus on two distinct desert regions that differ in ecology: (I) the Jizan desert in Saudi Arabia and (II) the Sahara in Algeria. To get acces to these regions we established partnerships with local research teams (Dr. Said Amrani, University of Science and Technology Houari Boumediene Algeria and. Dr. Heribert Hirt, KAUST, Saudi Arabia). Additionally, Dr. Ton Bisseling has a visiting professorship at King Saud University (KSU) in Riyadh, Saudi Arabia.

Exploiting the rhizosphere microbiome of native plant species in desert habitats

The Sahara is extremely dry and low in nutrients. The vegetation is dominated by Zygophyllaceae (e.g. Fagonia glutinosa, Peganum harmala, Tribulus teresteris and Zygophyllum album) and some legume species (Acacia trees and Retama shrubs). In contrast to the Sahara, the atmosphere in Jizan desert is relatively humid compared to the Sahara due to coastal fog, the soil is also extremely low in nutrients. Further, agricultural activities have been employed in Jizan. However, due to constant irrigation these fields salinified and became unsuited for further cultivation. These salinified agricultural fields are scarcely populated with only a single species, the legume Indigofera argentea. This pioneer vegetation species forms plots that only at later stages become intermingled with other species. Given the extreme environmental settings in both deserts, Sahara and Jizan, we hypothesise that the seed and root microbiomes play a pivotal role in conferring abiotic stress tolerance to the plant species growing under these harsh conditions. This hypothesis is supported by a pilot experiment in which we investigated the role of individual bacteria isolated from the I. argentea rhizosphere on Arabidopsis. Two (out of 30) bacterial species promoted plant growth in vitro under salt stress conditions, showing that the microbiome contains species that can contribute to stress tolerance. In this project we will exploit the spermosphere and rhizosphere of plant species native to the Sahara and Jizan deserts, aiming to identify and utilize microbial networks that can contribute to a broad-spectrum abiotic stress tolerance. To do so, we will focus on two Zygophyllaceae species; Fagonia glutinosa and Tribulus teresteris, respectively, both in Jizan as well as the Sahara. Furthermore, we will focus on two different Indigofera species: I. argentea that does grow in salinified agricultural fields as well in the surroundings, and its close relative Indigofera oblongifolia that is only present in the surrounding natural vegetation. We collected seeds of these 4 species and started selfing programs by single seed descent to create homozygous lines. Additionally, the transcriptome of the plant root under a range of growth conditions has been obtained, providing a reference transcriptome for RNA-seq based expression studies.

Major aims of this project

  1. Characterize the seed and root microbiomes of Fagonia glutinosa, Tribulus teresteris, Indigofera argentea and I. oblongifolia grown in their natural, desert habitats.
  2. Develop & apply a quantitative assay to monitor microbial effects on seed germination and abiotic stress tolerance of plant seedlings and subsequent growth (together with projects 1 and 2).
  3. Identify and reconstruct core microbial networks (bacteria and fungi) that can be applied to a broad spectrum of plant species to promote plant performance under abiotic stress conditions (together with projects 1 and 2).

Research plan

Workpackage 3.1. Characterization of spermo- and rhizospheres of desert vegetation

  • The rhizosphere microbiomes will be determined for two Indigofera species in Jizan (I. argentea and I. oblongifolia) and 2 Zhygophyllaceae species in Jizan as well as the Sahara (F. glutinosa, T. teresteris). To characterize the rhizosphere, we will collect roots of 10 individual plants (10 replicates) at each location. In case of I. argentea we will sample plants that grow at the salty agricultural fields, as well as at the neighbouring native habitat. Material will be collected in March 2015, the “optimal” growth season, and in mid-summer, the period with most severe abiotic stress. Additionally, soil samples will be collected at each location to determine nutrient composition as well as to preserve the soil microbiome for further studies.
  • To characterize the spermosphere, we will collect dispersed seeds at the growth season of the plants. As the natural growth season is more predictable for Jizan desert, we will focus these studies on this location.
  • Bacterial and fungal communities in soil, at the root and seed-soil interface (epiphytes) and inside roots and seeds (endophytes) will be characterized by 16S/18S rRNA sequencing, in close collaboration with partner I. Protocols will be applied similar as described for project 1. These analyses will provide insight which microbes are prominent in the spermo- and rhizospheres of the different plant species when compared to soil samples. This will reveal for each species the core and accessory microbiomes on seeds and roots.
  • We hypothesize that I. argentea, at least in part, exploits its root microbiome to tolerate high salt concentrations in some habitats. To get insight in which microbes may contribute to this character we will compare the root microbiome of I. argentea plants grown in salty agricultural fields with the microbiome of I. argentea and I. oblongifolia grown in natural desert habitat. We will store the meta-microbiome samples in glycerol to isolate the species/strains that make up the core microbiome of drought/salt tolerant plants.

Workpackage 3.2. A quantitative assay to monitor microbial effects on abiotic stress tolerance

  • To be able to conduct quantitative studies, a growth assay will be used that enables us to monitor plant performance under abiotic stress conditions (drought, heat, salinity, nutrient limitations) in response to microorganisms. To this end, plant growth, leaf surface and photosynthetic performance, and seed production will be assessed using non-destructive digital phenotyping.
  • We will first design a controlled growth system for selected desert plant species that can be used as experimental system in the lab (F. glutinosa, T. teresteris, I. argentea and I. oblongifolia). In Jizan we previously determined the nutrient content, which showed to be extremely low (e.g. nitrogen below detection level and phosphate ~3 mg/kg), whereas the salt concentration in the salty agricultural fields is between 100-150 mM NaCl. We will characterize the nutrient composition and water content of the soil in Sahara sand in a similar way. Furthermore, humidity and light intensity will be determined in the field. This information will be used to reconstruct these growth conditions in the lab (growth cabinet). For this, a sand culture system will be used that is already operational for Indigofera sp. This system enables us to monitor seed vigor, seedling behaviour and plant growth. To recreate the microbiome as found in natural habitats we will use sand collected at native sites and mix it with sterilised commercial sand in a ratio of 1:10. In control experiments, the desert sand will be sterilized by gamma-irradiation. To mimick saline growth conditions NaCl can be added along a concentration gradient. The microbial communities at the seed- and root-soil interface (epiphytes) and within the seed and root (endophytes) will be characterized by 16S/18S rRNA sequencing. Since the desert plant species are perennials, we will monitor their phenotype as well the rhizosphere during a 1-year period. Material (3 pots/replicates per time point) will be harvested and analysed after 1 week, and 1, 3, 6 and 12 months. Root microbiomes will be collected and analysed according to the protocols and methodologies described in project 1. These studies will reveal insight whether the core microbiome as found in native conditions can be reconstructed in the lab, and to what extend it contributes to the abiotic stress tolerance of of desert plants.

Workpackage 3.3  Identification of microbes that promote growth under abiotic stress conditions

  • We will use the experimental system setup as described in project 3.2 for reverse engineering of microbiome populations. To this end, we aim to isolate and culture the top candidate strains identified in 3.2, and inoculate sterilized sand with different combinations of these strains or consortia, varying both the presence of strains and their frequency in the population (see also project 3.1). A mathematical relation found between population structure and abiotic stress tolerance can be “inverted” to determine which composition changes are most likely to influence tolerance and are therefore most likely to yield informative results in a reverse approach. This is a form of active learning, widely studied in the machine learning community and already successfully applied in the life sciences (Danziger et al., 2007; Van den Berg et al., 2014). Plants will be grown under conditions that mimic the field conditions and the reconstructed microbiome will be analyzed and related to measured phenotype. Those microbes, or combinations of microbes, that have proven effect on the abiotic stress tolerance of the desert plant species will be tested also on crop plants. The selection of which crops will be used will be done in concordance with the industrial partners involved. For each crop species, the growth conditions will be adapted, such that crop species can grow, but still experience abiotic stress. Also we will include Piriformospora indica and soil bacterium Achromobacter piechaudii as positive controls in these experiments. These studies will provide insight in the utilization potential of the newly identified microbes.

 

II. UTILISATION PLAN

 

Problems

Crops have to resist abiotic stress factors like drought, heat and salinity and it is envisaged that this problem will become worse due to climate change. Hence, there is an urgent need for new sustainable measures that promote plant growth and protect plants against such abiotic stresses. Additionally, to meet the world’s future food demands, increased crop yields will have to be achieved also under sub-optimal growth conditions. To-date, however, there is only a limited understanding of the contribution of the seed and root microbiomes to the abiotic stress tolerance of plants.

Solutions and economic value

Deciphering the mechanisms affecting the seed and root microbiomes in relation to plant health under abiotic stress requires carefully controlled studies with relative simple systems. The desert ecosystems we aim to use in this project, in combination with reconstruction experiments under controlled laboratory conditions provide an unprecedented resolution in unravelling root microbiome functioning. We hypothesize that the effect of the spermosphere and rhizosphere on abiotic stress tolerance of plants is most prevalent on those plant species that grow in habitats with harsh conditions like natural deserts. Pilot studies of the applicant have shown that individual rhizosphere bacteria isolated from desert plant species (e.g. Indigofera) can promote plant growth of Arabidopsis and Indigofera plants under stress conditions. This project further builds on these initial findings and will focus on the characterization of distinct and novel microbial communities that promote plant tolerance to different abiotic stresses; including drought and salinity. The effect of these microbial communities will be studied on several field crops (e.g. onion, maize and soybean). The selected crops are of high socio-economic value worldwide and of specific financial interest to the companies contributing to this project.

Translation and implementation

Similar to project 1, utilization of the results into practical applications requires knowledge, expertise, materials and practical input of the companies in: a) seeds of crop species, b) seed technology & coatings, c) small-scale greenhouse and field trials, d) biotechnological use of microbes, e) fermentation, formulation, registration and marketing of microbial inoculants. Except for registration, formulation and marketing, many of the steps towards new products and technologies can be achieved within the proposed time-frame of 6 years.