Novel rapid screening assay to incorporate complexity and increase throughput in early-stage plant biological testing

Abstract

The search for new biological products with a positive impact on crop performance is typically initiated by laboratory based in vitro assays. However, live plants and their associated microbes are often removed from in vitro testing assays as a way to reduce biological complexity (variation) and facilitate molecular techniques in the pursuit of uncovering mode-of-action (MoA) mechanisms. Nevertheless, when studying biological candidates intended for use in agriculture, it is essential to incorporate this complexity and validate mechanisms under conditions as close to in situ as possible in order to understand the capacities and MoA of the biologicals in the intended application environments. To address this paradox, we have developed a high-capacity early-stage plant assay that incorporates a live non-sterile plant while also enabling molecular MoA investigations, and that can be conducted in laboratories without greenhouse facilities. The high-capacity design features plants grown in 8-chamber transparent boxes to allow for multiplex imaging and increased biological replicates for greater statistical power. The transparent box design allows the visualization of shoots, roots, tagged-microbes, or visible substrates, and further non-destructive access to shoots or roots for sampling. The boxes are held in racks that hold eight plant boxes during growth in a 19 by 17 cm space, further increasing the throughput to >670 plants per m² and easing the logistical challenges of plant assays. Furthermore, the box can support various levels of microbial complexity with the option to select the plant growth medium that meets experimental objectives, as well as using sterile or non-sterile seeds. A script-based post-imaging quantification was developed to automate image processing and allow for individual plant readings, further enabling increased statistical confidence. As proof of concept, we use the high-capacity plant system to evaluate the biocontrol potential of Pseudomonas protegens and the biostimulation potential of Pseudomonas koreensis, and are in both cases able to show statistically significant differing plant biomass between treatments under these closer-to-nature conditions. We further demonstrate that the high-capacity plant system is suitable for paired molecular investigations by performing metabolomics and qPCR DNA quantification directly from the plant box to explore in situ chemical MoA, as well as confirm the survival of the P. protegens strains to validate their role in the improved plant phenotype. In conclusion, the study presents a modular high-capacity plant assay system that enables increased throughput functional testing of microbial biocontrol and biostimulant candidates in planta. This novel assaying system saves time, reduces human error, provides quantitative and non-destructive in planta data, and can be used in laboratories without greenhouse facilities. We therefore believe it provides a potent early-stage testing option that bridges in vitro and greenhouse testing, and will expedite the discovery of superior next-generation biological products in agriculture.