Rhizoboxes offer the advantage of localized sampling in soil using sorption media such as paper and membrane filters, compound specific ion exchange binding resin or micro-suction cups placed closed to root zones of inTherest to collect exudates . Moreover, in a rhizobox fitted at the bottom with a porous rootimpenetrable membrane, a root mat is allowed to be formed which is then further transferred onto a collection compartment . The collection compartment containing soil could then be cut into thin slices parallel to the membrane to represent differing distances from the rhizosphere . While this approach can be used to investigate exudate release and sorption under soil conditions, the root mat growth generalizes exudate production in terms of the whole root system and occludes spatial exudation patterns. In a hybrid set up by Oburger et al. , the rhizobox is transplanted to a second specialized rhizobox for continued vertical root growth. This specialized rhizobox consists of a nylon membrane close to the transparent side to restrict root growth into the soil except for root hairs . This creates a vertical flat root mat onto which localized exudate samples can be collected. A comparison of this novel set up to conventional collection methods showed that amino acid exudation rates were most varied among the different methods , further highlighting the need for specialized chambers. Nonetheless, successful implementation of these chambers is still limited to fast-growing plants which can form active root mats. The high density of root mats could also lead to unnatural root exudate levels and an overestimation of rhizosphere effects. In addition, care has to be given to the choice of membrane as selective sorption of certain root exudates onto the membrane may also occur . Free-living nematodes are ubiquitous in the soil. They are beneficial to the plants by playing a role in nutrient cycling and in defense against insects and microbial infections through signaling interactions with the roots .
Conversely, infections by parasitic nematodes in the roots increase the plant’s susceptibility to stress and other pathogenic bacteria, fungi,growing hydroponically and viruses creating major losses in crop productivity . With an impending rise in nematode infections due to climate change, understanding nematode behavior and interactions in the rhizosphere becomes important to develop appropriate biocontrol methods to ensure long term food security . Traditional nematode studies are performed in petri dishes with agar or culture media . However, these substrates do not accurately emulate the physical textures and heterogeneity of soil and create homogenous solute and temperature gradients which could impact nematode behavior and interactions with the roots . Indeed, nematode motility speed and dispersal decreased in substrates more closely mimicking sand . On the other hand, studying nematode behavior in the soil is a difficult endeavor as its near-transparent body and small size makes it almost indistinguishable from soil particles. Cross-sectioning and staining infected roots make it possible for nematode visualization but they are destructive and provide only static snapshots of cellular changes or nematode behavior during infections . On the other hand, microscopy rhizosphere chambers provide non-invasive detection and observation of nematode activity in the rhizosphere . The roots in these chambers grow between a glass slide and a nylon membrane . The membrane restricts movement of roots except root hairs into the soil while the transparent glass enables microscopy of the roots at high resolution . Coupled with fluorescently stained nematodes, microscopy rhizosphere chambers allowed for non-destructive in situ observations of nematode infection in its host species over the entire life of the parasite . Nonetheless, staining nematodes is an additional challenge as nematode cuticles are impermeable to stains . This can, however, be alleviated by using advanced imaging technologies which eliminates the need for staining.
A recent study demonstrated live screening of nematode-root interactions in a transparent soil-like substrate through the use of label-free light sheet imaging termed Biospeckle Selective Plane Illumination Microscopy coupled with Confocal Laser Scanning Microscopy . Using this set up, researchers were able to monitor roots for nematode activity at high resolution and suggest its possible use in rapid testing of chemical control agents against parasitic nematodes in soil-like conditions . Fungal communities in the rhizosphere are involved in the degradation of organic matter in the soil and subsequent nutrient turnover affecting plant health as well as the microbial community . Fungal biomass often reaches a third of total microbial biomass carbon and almost all terrestrial plants are able to form symbiotic associations with mycorrhizal fungi . The majority of these associations are with arbuscular mycorrhiza fungi which penetrate into root cortex cells to form highly branched structures . The investment of photosynthetic carbon by plants to AMF is rewarded with increased nutrient availability made possible by the extended hyphal network in the soil. For instance, up to 90% of phosphorus uptake in plants can be contributed by symbiosis with AMF . AMF networks in the soil also influence water retention and soil aggregation further impacting plant growth . Moreover, next-generation sequencing technologies and advances in imaging techniques have greatly improved our knowledge on the taxonomical and functional properties of fungal communities in the rhizosphere . However, these methods are optimized for fine scale analysis and are not capable of assessing the foraging capabilities of hyphal networks which can span across centimeter to meter scales. Toward this end, several researchers have used compartment setups with physical barriers created by 20–37 µm nylon membranes which restrict movement of roots but not mycorrhizal fungi. This separation creates root-free and plant free soil compartments connected only by mycorrhizal fungi to examine the transport of various compounds across these compartments. Using this set up, the importance of mycorrhizal fungi in the flow of different elements such as carbon , nitrogen and phosphorus between plants, soil and microbes over centimeter distances have been validated.
Repeated disruption of the hyphal connections also led to a decreased resistance in plants to drought stress . The membranes can also be placed horizontally to create different depth gradients to investigate hyphal contributions to water uptake . In some studies, an additional 1.5–3 mm air gap is created between two membranes with a wire net to restrict solute movement between two chambers . A common feature of these set ups is the size-exclusion membranes which proved to be critical in distinguishing fungal hyphae processes in the rhizosphere soil. In addition to AMF interactions, a split root set up, which separates the roots of one plant into halves, can be introduced to investigate the systemic response of plants . In essence, the split-root system directs the growth of the roots to generally two different growth conditions and enables the investigation of whether a local stimuli have a local or global response which can be observed at the root or shoot level . Split-root systems are widely studied and have been adapted to rhizoboxes as well as to pots and tubes . In the rhizosphere, plants host a wide diversity of bacteria on the surface of the root as well as within roots in the vascular tissues . Due to its abundance and importance,ebb flow tray the bacterial community in the rhizosphere is perhaps the most widely studied among other microbial members in the rhizosphere ecosystem. While the study of endophytic bacteria requires inevitable destructive sampling due to its localization, several non-destructive approaches have been developed to study microbes inhabiting the rhizoplane. One of the most widely studied plant-microbe interactions in the rhizosphere is that of the symbiotic relationship between legumes and rhizobia . Once a potential nodule forming bacteria is isolated, it is often required to authenticate its nodule forming phenotype by inoculating on host plants. However, conventional methods such as the use of soil pouches do not allow long term incubation, while “Leonard jars,” consisting of two stacked glass jars forming the top soil layer and the bottom nutrient solution layer, can be expensive and time consuming . A recent study challenges this by describing the use of clear plastic CD cases as minirhizotrons with potential for use in phenotyping root traits such as legume formation, and demonstrated innovation that democratizes research opportunities in rhizosphere research . Other microbial interactions in the rhizosphere, however, may not result in visible changes to the root system and often rely on next-generation omics technologies. As such, physical separation of the rhizosphere from the bulk soil becomes paramount in elucidating changes to microbial community and interactions. One approach to this end is the use of nylon bags with differing pore sizes . The nylon bag restricts the movement of roots and the soil inside the bag is then regarded as the rhizosphere soil to compare against the surrounding root free bulk soil . Developing further on this concept, Wei et al. designed a specialized rhizobox that allowed repeated non-destructive sampling by adding individual nylon bags of root-free soil surrounding the root compartment which are then used as a proxy for the rhizosphere . These methods allowed easy distinction of the rhizosphere and the bulk soil but, we now know that the rhizosphere community is not only distinct from the bulk soil but also varies with type, part and age of the root, largely as a consequence of varying root exudation patterns . Studying this phenomenon in situ in the soil requires separation of desired roots from others without disturbance to plant growth or soil. To address this, researchers have used a modified rhizobox design with a side compartment to regulate root growth and quarantine specific roots from the main plant chamber . This additionally creates easy distinction between old and new roots and allows testing on specific quarantined roots despite plant age. A study using this set up showed specific microbial chemotaxis toward different exudates on an individual root whereas another showed spatial and temporal regulation of niche differentiation in microbial rhizosphere guilds .
Similar physical perturbations to regulate root growth in response to microbial stimuli have also been applied in the microscale and are explored in the next section. Our assessment of the major growth chambers showed that most of the systems applied share similarities in basic structural components such as in the use of two parallel sheets in rhizobox based devices. While these growth chambers brought many of the rhizosphere processes to light, limitations do exist. One limitation is with the scale of applicability. Most of these growth systems are mesoscale and can easily reproduce potscale studies but may not be easily translatable to interactions occurring at the microscale nor recapitulate processes occurring at field-relevant scale. The next section describes advances in technology resulting in a new wave of unique devices making use of microfluidic processes and fabricated ecosystems which are specifically made to investigate specific rhizosphere processes.A complex web of biochemical processes and interactions occur in microscale dimensions in the rhizosphere. Having the ability to interrogate and manipulate these microscale processes and environmental conditions with high spatiotemporal resolution will elucidate mechanistic understanding of the processes. Microfluidics has proven to be a powerful approach to minimize reagent usage and to automate the often-repetitive steps. The microscale of the channels also allows precise control of reproducible conditions utilizing the laminar flow and automated fluidic operations . In addition, the microfluidic devices are well integrated with conventional imaging techniques by using a glass slide or coverslip as a substrate bonded with polydimethylsiloxane . These characteristics, as well as the ability to rapidly prototype and reproducibly manufacture using soft lithography technique, have enabled new ways of interrogating and studying the rhizosphere environment in a reproducible manner.They have an opening port, sometimes with pipette tips inserted into the PDMS body where the seed of the seedling rests and a microchannel where the primary root grows into. The dimension of the channel depends on the type and age of the plant. For example, an Arabidopsis thaliana’s seedling is typically grown in a microfluidic device up to 10 days, with chamber dimension around 150 to 200 µm in height, whereas the Brachypodium distachyon seedling chamber is 1 mm in height due to its thicker roots . Media and/or inoculation of the microbiome is achieved through additional channels to the main chamber. The PDMS body with the channels is typically bonded on a 50 mm by 75 mm microscope slide, and is made to accommodate multiple plants to increase throughput.