Abstract
Free living nematodes (FLN) are microscopic worms found in all soils. While many FLN species are beneficial to crops, some species cause significant damage by feeding on roots and vectoring viruses. With the planned legislative removal of traditionally used chemical treatments, identification of new ways to manage FLN populations has become a high priority. For this, more powerful screening systems are required to rapidly assess threats to crops and identify treatments efficiently. Here, we have developed new live assays for testing nematode responses to treatment by combining transparent soil microcosms, a new light sheet imaging technique termed Biospeckle Selective Plane Illumination Microscopy (BSPIM) for fast nematode detection, and Confocal Laser Scanning Microscopy for high resolution imaging. We show that BSPIM increased signal to noise ratios by up to 60 fold and allowed the automatic detection of FLN in transparent soil samples of 1.5 mL. Growing plant root systems were rapidly scanned for nematode abundance and activity, and FLN feeding behaviour and responses to chemical compounds observed in soil-like conditions. This approach could be used for direct monitoring of FLN activity either to develop new compounds that target economically damaging herbivorous nematodes or ensuring that beneficial species are not negatively impacted.
Highlights
Free living nematodes (FLN) are microscopic worms that live in the soil
The use of glass plates was designed to allow both upright imaging of the roots with light sheet or biospeckle, and in a horizontal position for use in confocal laser scanning microscopy
In experiment 4, we showed that Confocal Laser Scanning Microscopy (CLSM) allows high resolution imaging of the nematodes that are detected with Biospeckle Selective Plane Illumination Microscopy (BSPIM) (Fig. 6)
Summary
Free living nematodes (FLN) are microscopic worms that live in the soil. They move freely through soil making use of the thin film of water surrounding the soil particles. The study of nematode behaviour could benefit greatly from live imaging systems if detection could be made in a substrate that mimics soil Light sheet based techniques such as SPIM (Selective Plane Illumination Microscopy) have shown great potential for live imaging studies[24,25,26,27,28], but light scattering itself may not allow easy separation of nematode and root in the image[29]. We have assessed the ability of various techniques to 1) culture plants and nematodes in soil-like conditions while allowing easy introduction of chemical controls; 2) rapidly locate and map the distribution of live nematodes in the rhizosphere of a growing root in 3D; 3) quantify infestation levels; and 4) image nematode behaviour in high resolution. We propose a general framework to assess FLN risk to plant health and test the efficacy of chemical controls
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