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The root-associated microbiome (rhizobiome) affects plant health, stress tolerance, and nutrient use efficiency. However, it remains unclear to what extent the composition of the rhizobiome is governed by intraspecific variation in host plant genetics in the field and the degree to which host plant selection can reshape the composition of the rhizobiome. Here, we quantify the rhizosphere microbial communities associated with a replicated diversity panel of 230 maize ( Zea mays L .) genotypes grown in agronomically relevant conditions under high N (+N) and low N (-N) treatments. We analyze the maize rhizobiome in terms of 150 abundant and consistently reproducible microbial groups and we show that the abundance of many root-associated microbes is explainable by natural genetic variation in the host plant, with a greater proportion of microbial variance attributable to plant genetic variation in -N conditions. Population genetic approaches identify signatures of purifying selection in the maize genome associated with the abundance of several groups of microbes in the maize rhizobiome. Genome-wide association study was conducted using the abundance of microbial groups as rhizobiome traits, and n=622 plant loci were identified that are linked to the abundance of n=104 microbial groups in the maize rhizosphere. In 62/104 cases, which is more than expected by chance, the abundance of these same microbial groups was correlated with variation in plant vigor indicators derived from high throughput phenotyping of the same field experiment. We provide comprehensive datasets about the three-way interaction of host genetics, microbe abundance, and plant performance under two N treatments to facilitate targeted experiments toward harnessing the full potential of root-associated microbial symbionts in maize production. 

Although there have been numerous studies describing plant growth systems for root exudate collection, a common limitation is that these systems require disruption of the plant root system to facilitate exudate collection. Here, we present a newly designed semi-hydroponic system that uses glass beads as solid support to simulate soil impedance, which combined with drip irrigation, facilitates growth of healthy maize plants, collection and analysis of root exudates, and phenotyping of the roots with minimal growth disturbance or root damage.

This system was used to collect root exudates from seven maize genotypes using water or 1 mM CaCl₂, and to measure root phenotype data using standard methods and the Digital imaging of root traits (DIRT) software. LC–MS/MS (Liquid Chromatography—Tandem Mass Spectrometry) and GC–MS (Gas Chromatography—Mass Spectrometry) targeted metabolomics platforms were used to detect and quantify metabolites in the root exudates. Phytohormones, some of which are reported in maize root exudates for the first time, the benzoxazinoid DIMBOA (2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one), amino acids, and sugars were detected and quantified. After validating the methodology using known concentrations of standards for the targeted compounds, we found that the choice of the exudate collection solution affected the exudation and analysis of a subset of analyzed metabolites. No differences between collection in water or CaCl₂were found for phytohormones and sugars. In contrast, the amino acids were more concentrated when water was used as the exudate collection solution. The collection in CaCl₂required a clean-up step before MS analysis which was found to interfere with the detection of a subset of the amino acids. Finally, using the phenotypic measurements and the metabolite data, significant differences between genotypes were found and correlations between metabolites and phenotypic traits were identified.

A new plant growth system combining glass beads supported hydroponics with semi-automated drip irrigation of sterile solutions was implemented to grow maize plants and collect root exudates without disturbing or damaging the roots. The validated targeted exudate metabolomics platform combined with root phenotyping provides a powerful tool to link plant root and exudate phenotypes to genotype and study the natural variation of plant populations.

 

Root exudates are important for shaping root-associated microbiomes. However, studies on a wider range of metabolites in exudates are required for a comprehensive understanding about their influence on microbial communities. We identified maize inbred lines that differ in exudate concentrations of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and γ-aminobutyric acid (GABA) using a semi-hydroponic system. These lines were grown in the field to determine the changes in microbial diversity and gene expression due to varying concentrations of DIMBOA and GABA in exudates using 16S rRNA amplicon sequencing and metatranscriptomics. Results showed individual and interaction effects of DIMBOA and GABA on the rhizosphere and root endosphere β-diversity, most strongly at the V10 growth stage. The main bacterial families affected by both compounds were Ktedonobacteraceae and Xanthomonadaceae. Higher concentrations of DIMBOA in exudates affected the rhizosphere metatranscriptome, enriching for metabolic pathways associated with plant disease. This study validated the use of natural variation within plant species as a powerful approach for understanding the role of root exudates on microbiome selection. We also showed that a semi-hydroponic system can be used to identify maize genotypes that differ in GABA and DIMBOA exudate concentrations under field conditions. The impact of GABA exudation on root-associated microbiomes is shown for the first time.

 

ABSTRACT Root-associated microbes are key players in plant health, disease resistance, and nitrogen (N) use efficiency. It remains largely unclear how the interplay of biological and environmental factors affects rhizobiome dynamics in agricultural systems. In this study, we quantified the composition of rhizosphere and bulk soil microbial communities associated with maize ( Zea mays L.) and soybean ( Glycine max L.) in a long-term crop rotation study under conventional fertilization and low-N regimes. Over two growing seasons, we evaluated the effects of environmental conditions and several treatment factors on the abundance of rhizosphere- and soil-colonizing microbial taxa. Time of sampling, host plant species, and N fertilization had major effects on microbiomes, while no effect of crop rotation was observed. Using variance partitioning as well as 16S sequence information, we further defined a set of 82 microbial genera and functional taxonomic groups at the subgenus level that show distinct responses to treatment factors. We identified taxa that are highly specific to either maize or soybean rhizospheres, as well as taxa that are sensitive to N fertilization in plant rhizospheres and bulk soil. This study provides insights to harness the full potential of soil microbes in maize and soybean agricultural systems through plant breeding and field management. IMPORTANCE Plant roots are colonized by large numbers of microbes, some of which may help the plant acquire nutrients and fight diseases. Our study contributes to a better understanding of root-colonizing microbes in the widespread and economically important maize-soybean crop rotation system. The long-term goal of this research is to optimize crop plant varieties and field management to create the best possible conditions for beneficial plant-microbe interactions to occur. These beneficial microbes may be harnessed to sustainably reduce dependency on pesticides and industrial fertilizer. We identify groups of microbes specific to the maize or to the soybean host and microbes that are sensitive to nitrogen fertilization. These microbes represent candidates that may be influenced through plant breeding or field management, and future research will be directed toward elucidating their roles in plant health and nitrogen usage.