Exploring Mycorrhizosphere Microbial Diversity in Sterilized Rhizobox Systems with Third-Generation Sequencing

The mycorrhizosphere is a highly dynamic soil zone characterized by intense interactions among plant roots, arbuscular mycorrhizal fungi (AMF), and associated microbial communities. This biologically active region is pivotal in nutrient cycling, root colonization, and soil sustainability. Despite its ecological importance, the mycorrhizosphere remains poorly understood due to its challenging accessibility. Conventional destructive sampling methods often fail to isolate this specific zone, leading to a limited understanding of its microbial composition. To address these challenges, we employ rhizobox systems designed to maintain the integrity of the mycorrhizosphere. These systems allow precise sampling of the region directly influenced by root exudates and AMF activity. By consistently accessing the zone of inoculation, the rhizobox enables repeated observations and measurements, enhancing our ability to study the microbial dynamics of this elusive soil environment. This study aims to uncover key taxonomic groups within the mycorrhizosphere, focusing on identifying mycorrhiza-helper bacteria (MHB) and fungi (MHF) that could improve mycorrhizal colonization and soil health. The primary objectives of this research are to: i) Characterize Microbial Diversity: Third-generation sequencing technologies (MinIon R10.4.1 V14, Nanoporetech) will be used to profile bacterial and fungal communities within the mycorrhizosphere of Orchard Baby maize (Zea mays). The focus will be on taxonomic identification using genetic markers such as 16S rRNA for bacteria and ITS for fungi; ii) Establish a Controlled Environment: Sterilized rhizobox systems filled with an artificial soil mixture will serve as the experimental framework. Microbial inocula derived from native forest soils, conservation-managed agricultural soils, and intensively managed agricultural soils will be applied in dilution series (10⁻¹, 10⁻³, and 10⁻⁶) to mimic natural microbial gradients. iii) AMF Inoculation: Rhizophagus irregularis (syn. Glomus intraradices), a well-documented AMF species, will facilitate root colonization and provide a controlled environment to evaluate microbial interactions. The study combines molecular sequencing with temporal sampling to examine microbial recruitment by mycorrhizal plants. DNA extraction will occur at three stages (beginning 1 month and 3 months after the start) to capture temporal changes in microbial composition. The focus will be on identifying the dominant taxa in the mycorrhizosphere. Our project seeks to provide the first high-resolution taxonomic profiles of microbial communities in the mycorrhizosphere. We also expected to establish a methodological framework for studying elusive soil zones with high biological activity and to highlight the microbial taxes associated with AMF colonization. Integrating rhizobox systems with advanced sequencing will provide insights into microbial dynamics that can inform sustainable agricultural practices, particularly in enhancing nutrient uptake and soil resilience.