New live screening of plant-nematode interactions in the rhizosphere

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.

Figure 1

Imaging systems for the detection of nematode activity. (a) Plants and nematodes cultured in chambers created by two microscope slides separated by a spacer, between which transparent soil (TS) was held as the growth medium. The chambers were scanned by a laser light sheet either to detect the numbers of nematodes present or to observe their activity within the rhizosphere. Two scanning modes were used: (b) nematode automated detection was carried out using a light sheet of 1 mm in width moved by increments of 125 µm (an area of overlap, not shown, is created which prevents small or mobile nematodes being missed). (c) 3D rendering was carried out with light sheets of 30 µm in width by increments of 125 µm. (d) Principle of Biospeckle Selective Plane Illumination Microscopy (BSPIM) Imaging. The illumination is composed of a laser light source and two cylindrical lenses. Light from a laser (I) is first passed through an adjustable slit (II1) before being vertically broadened by a cylindrical lens (II2) to produce the light sheet. A second cylindrical lens (II3) is used to focus the light sheet on the sample (III), which is translated along the z-axis by a motorised stage (IV). Images were collected by stereomicroscope and camera (V-VI) perpendicularly to the sheet of light while using a polariser (VII) to remove reflected light. Image sequences were subsequently analysed for dynamic scattering (VIII) resulting in a map of biospeckle intensity for each step moved by the sample.

Figure 2

Optimisation of transparent soil microcosms. (a) Lettuce grown for 8 days in transparent soil growth chambers consisting of two double-size microscope slides separated by a silicone ring. Plants are shown before and after immersion in refractive index matched Ludox® TMA. (b) Bubbles were removed from saturated transparent soil by suction, resulting in a significant decrease in the number of bubbles per cm². (c) Fluorimeter cuvettes filled with transparent soil saturated in water and refractive index matched Ludox® TMA, Percoll® and trehalose. A line has been drawn behind the cuvettes for comparison of optical clarity. (d) Quantitative assessment of optical clarity of the substrates shown in (c). Measurements were made along 4 cross sections; these ran perpendicularly to the black line behind each sample and are shown in red in (c). Top left, close-up view of the samples at cross section 3. Top right, grey values of the image along cross section 3. Because grey values have black as their lowest intensity, grey values drop at the point where cross sections pass the black line. As the black line is clearer the better the refractive indices are matched, a larger drop in grey values indicates greater transparency. Below are shown the mean difference between grey values for noise and signal (i.e. the minimum grey value). Values are averaged across the 4 sections and show that refractive index matching with the colloids Ludox® TMA and Percoll® achieved similar transparency to that of the trehalose solution.

Figure 3

Nematode motility and plant germination rate. (a) Comparison of refractive index matching liquids Ludox® TMA, Percoll® and trehalose with water as a germination and growth medium for plants in transparent soil. Columns represent the average count within replicate groups of 5, with error bars representing the standard deviation. (b) Survival of soil FLN in transparent soil with Percoll®, Ludox® TMA, trehalose and dazomet dissolved in Ludox® TMA at 100 mg L⁻¹. Each treatment was tested on 5 microcosms of transparent soil containing 5 nematodes each. For each microcosm, the percentage of moving nematodes was calculated as a percentage of all visible nematodes. The figure shows the range of percentages across all 5 microcosms, with boxes representing 50% of observations and whiskers representing x 1.5 the interquartile range. Outliers outside x 1.5 and 3 the interquartile range are represented by an open circle and asterisks respectively. Spontaneous movement over the first 24 hours was visible in all samples for Percoll®, Ludox® TMA and trehalose but not in the presence of dazomet. After 1 week, less activity was also observed in trehalose. Spontaneous movement declined in the presence of dazomet within 24 hours and was completely absent after 2 weeks.

Figure 4

Correlations of numbers of inserted nematodes with mean numbers of nematodes detected by biospeckle, with 3 replicates for each data point and error bars representing the standard deviation. Images of biospeckle activity were obtained at different depth within the sample using the GD method. Images obtained with the GD method at different depths were stacked to constitute a volume image. The volume image was then filtered, and the number of areas with high activity detected automatically using ImageJ 3D object counter. (a) Nematodes detected while in suspensionin a Ludox® TMA solution. Black markers represent live nematodes, open red markers represent nematodes killed by 90 seconds at 60 °C. (b) nematodes detected in transparent soil immersed in Ludox® TMA with 5 replicates for each data point and the standard deviation represented by the error bars. Red boxes show the region of interest containing the nematodes.

Figure 5

Two examples of bright field images (on the left) and images of the biospeckle activity obtained using the GD method (right) of FLN in the vicinity of lettuce roots grown in transparent soil lit by a green laser light sheet. Biospeckle from the root has been blotted out by saturation, allowing nematodes (circled in red) to be easily located. In comparison to bright field, biospeckle imaging provides enhanced contrast and the reduction of noise from scatter, visible as white pixels in the brightfield images. Superimposed green traces show the grey values of cross-sections through the images, shown as a white line. Grey values for bright field images had higher levels of variation caused by the scatter of laser light by the transparent soil environment and the root, visible here only in the bright field images. By comparison, this variation was greatly reduced in the biospeckle images.

Figure 6

CLSM imaging of plants and nematodes cultured in transparent soil saturated in Ludox® TMA. N. tabacum (a) and P. multiflora (b) stained with calcofluor and growing among transparent soil particles stained with sulforhodamine B. A plant-feeder (c) and a bacterivore (d) associated with roots of N.tabacum. Intestinal autofluorescence is visible in green in the bacterivore. Nematode head regions are indicated by red arrows.

Figure 7

Distinguishing nematodes from their surroundings in transparent soil with CLSM. (a) Unstained plant feeder among sulforhodamine B stained particles saturated in Percoll® and 0.1 g L⁻¹ calcofluor. (b) Autofluorescence in response to 488 nm laser irradiation of a nematode among sulforhodamine B stained transparent soil particles. (c) Autofluorescent nematode on calcofluor stained root with sulforhodamine B stained transparent soil particles. The nematode is indicated by the white arrow. (d) Intestinal autofluorescence in response to 488 nm (green) and 561 nm (red) laser irradiation. (e) Trichodorid with fluorescein in the intestine.

Figure 8

Framework for screening FLN risk to plant health and testing the efficacy of chemical interventions. (a) Rapid assessment of nematode population is first required to analyse the threat level. In order to accelerate the analysis, BSPIM can provide basic metrics on the nematode population, providing parameters such as abundance, motility and differential response to treatment. Standardised assays using substrate consisting of Ludox® TMA within a fluorescence cuvette will allow sample preparation and nematode detection in high throughput. (b) Once a threat is identified, identification of suitable treatment of the threat can be achieved by initial selection of the FLN of interest and the use of transparent soil assays, for example to test the efficacy and timing of treatment application. (c) Finally, detailed studies of nematode behaviour including responses to chemical treatment can be carried using transparent soil microcosms in which nematodes and plants can be co-cultured. Biospeckle (BSL) imaging and Confocal Laser Scanning Microscopy (CLSM) can be combined for efficient quantification of treatment effect on nematode behaviour.