Plant-parasitic nematodes (PPNs) especially root-knot (
Since data of laboratory bioassays can be extrapolated to understand the actual nematode behavior in natural soil and around the host root, researchers have frequently used agar and sand as assay media to test different hypotheses (Robinson, 2000; Spence et al., 2008; Farnier et al., 2012). Plenty of modifications were adopted for agar-and sand-based assays because both of them allow good nematode dispersion and stimulus diffusion (Rasmann et al., 2005; Ali et al., 2010). However, in the rigid agar medium nematodes move on their sides in two-dimensions which is quite unlike of their movement in natural soil environment. On agar surfaces nematodes can be trapped in water films. In addition, the opaque nature of sand renders the observation of nematode movement toward the concentration gradient of a test compound in sand column difficult (Spence et al., 2008). Of late, due to its non-rigid texture and high transparency, pluronic gel (PF-127) has emerged as an ideal medium to investigate the short-distance attraction of PPNs to host roots and their accumulation around certain sites (Wang et al., 2009a, 2009b, 2010; Dutta et al., 2011; Reynolds et al., 2011; Kumari et al., 2016; Dash et al., 2017). Nematodes move in three-dimensions in PF-127 medium which allows more realistic evaluations of nematode-host interactions.
Other three-dimensional behavioral assays include the use of micro-molded substrates (to study the effect of pore structure on nematode migration; Eo et al., 2007, 2008) and gel-filled micro channel array (
The extremely simple in vitro assays such as PF-127 assays provide sufficient traction to allow PPN movement toward a chemical gradient (Wang et al., 2009b, 2010). However, high spatial resolution (due to reduced convection) property of PF-127 gel sometimes results in tight clump formation by PPNs (Wang et al., 2009a, 2010) and numerous sinusoidal tracks of PPN movement on the gel surface (Fig. 1), which inhibits the quantitative analyses of PPN chemotaxis toward the concentration gradient of a test chemical. In order to overcome this problem, in the present study, agar was combined with PF-127 gel (poured in separate areas) in a petri plate. Test compounds were applied in the wells created in agar, adjacent to the interface with PF-127 gel. J2s of
A pure culture of
Seeds of tomato (cv. Pusa Ruby), tobacco (cv. Petite Hawana), and marigold (cv. Arpit) were surface sterilized and germinated via standard methods (Papolu et al., 2013; Dutta et al., 2015). Each of the five- to six-day-old seedlings was hydroponically grown in half strength Hoagland solution (Hoagland and Arnon, 1950) in 15 ml Falcon conical centrifuge tubes (Sigma-Aldrich) for 15 d followed by removal and insertion of the roots of the plantlets (post washing) in sterile distilled water in a fresh Falcon tube for another 5 d. The whole assembly (covered in aluminium foil) was maintained in a growth chamber at 28°C, 70% RH, and 14 hr: 10 hr light: dark photoperiod. Sterile water containing the root exudates were pooled from 50 plants for each of the hosts, filtered through Whatman No. 1 filter paper and concentrated to 5 ml suspension (from initial volume of 50 ml) for each of the hosts in Eppendorf tubes via vacuum evaporation in a Speed Vac Concentrator (Labconco) using a vacuum of 100 to 500 mTorr.
Pluronic F-127 (PF-127) (Sigma-Aldrich) gel was prepared as described previously (Wang et al., 2009a). To prepare 0.8% agarose gel, 0.8 g of agarose powder (Sigma-Aldrich) was dissolved in 100 ml of sterile distilled water in a microwave oven. Six ml of agarose gel was poured onto a 50×10 mm petri plate (Tarsons, part number - 461010) and allowed to solidify. Two parallel imaginary lines, each equidistant of 1.5 cm from the center of the petri plate, were drawn (Fig. 2). Agar was scooped out from the area between these two lines using sterilized forceps and was filled with 3 ml of 23% PF-127 gel and allowed to solidify at room temperature (i.e. >15°C). Small wells of 1.5 mm diameter were created (by scooping out the agar halfway through the plate with rear end of 1 ml pipette tip) on the agar surface adjacent to agar-PF-127 junction (almost 1 cm distant from the nearest edge of the petri plate) for application of test compounds (Fig. 2).
To quantitatively define the concentration gradient of a test compound in the assay plate, a colorimetric assay using acid fuchsin was carried out. PF-127 hardly adsorbs acid fuchsin which aids in rapid analysis of their concentration distribution in PF-127 gel by measuring the color intensity. Also, 20 µl of acid fuchsin (stock solution: 3.5 g acid fuchsin, 250 ml acetic acid, 750 ml distilled water) was pipetted into a well in assay plate and incubated at room temperature. After 40 min, 50 µl each of the test samples was collected from PF-127 gel at 2, 5, 10, and 15 mm distance from the application point of acid fuchsin and liquefied on ice. To each sample, distilled water was added to make up the volume to 1 ml for measurement of their absorbance at 550 nm in a standard spectrophotometer (Eppendorf Biophotometer Plus). Experimental samples were measured against the average of three controls containing PF-127 gel and distilled water. A standard curve was generated by plotting the various concentrations of acid fuchsin in PF-127 gel vs the absorbance measurements of different concentrations of acid fuchsin that were fit to a linear correlation model (
To measure the attraction or repulsion of J2 in response to test chemicals, various volatile compounds such as isoamyl alcohol, 1-butanol, benzaldehyde, 2-butanone, and 1-octanol (previously used in
In order to measure the attraction or repulsion of J2 in response to host root exudates, 10 µl of neat exudates of tomato, tobacco, and marigold were separately screened in the assay plate as described above. The diluent was water in this case.
All the experiments were carried out with at least three technical replicates and were repeated at least thrice. Data were initially checked for normality and compared using one-way analysis of variance with Tukey’s HSD tests in SAS statistical package. All individual treatments were statistically compared to negative controls, as stated in the figure legends.
We measured the change in color intensity of acid fuchsin stain in assay plate. Qualitatively, it was ascertained that acid fuchsin can diffuse through the PF-127 medium from higher concentration to lower concentration within 40 min to establish equilibrium (Fig. 4A). We estimated that the concentration gradient from the point source to nematode inoculation point remained in a steady state up to 4 hr. Acid fuchsin concentration measured (in terms of absorbance at 550 nm) at indicated distances from the point source suggested the gradual decrease of test compound concentration from the point source (Fig. 4B). This suggests that similar to acid fuchsin our assay plate could also establish the concentration gradient of different volatile and non-volatile test compounds. However, we assume that all of the volatile compounds may not behave the same since the chemical structure and molecular weight of volatiles differ considerably than that of acid fuchsin.
By contrast, upon 1-butanol exposure, attraction increased gradually from neat to 10−4 dilution, suggesting 1-butanol was attractive at lower concentrations. The CI was documented as 0.25 ± 0.1 (
Surprisingly, 1-octanol was not attractive at any of the concentrations and was repulsive at high concentrations. CI was observed as −0.1 ± 0.08 (
Photographs showed that in response to neat concentrations of isoamyl alcohol and 2-butanone exposure, greater number of J2s was accumulated at the agar-PF-127 junction at the odorant side and lesser number of J2s was accumulated at the agar-PF-127 junction at the diluent side (Fig. 5B).
The diluted host root exudates did not elicit any significant chemotactic response of
Compared to the free-living worm,
One of the major technical issues with agar plate assays is that nematodes venture around both on the surface of the gel and within the gel matrix. This makes it difficult to track and quantify the nematode behavior accurately. Although PF-127 gel allows nematode movement in three-dimensional plane, same is true for PF-127-based assays as well (Hida et al., 2015). In line with the
We confirmed experimentally the concentration gradient formation of a chemical compound in our assay plate. This was achieved by monitoring and quantifying the changes in the color intensity of acid fuchsin over time from the point source in PF-127 medium. We assume that different volatile and non-volatile test compounds may establish concentration gradient in our assay plate. However, this may not hold true for all the volatile compounds because chemical structure and molecular weight of volatiles differ considerably than that of acid fuchsin. It has been suggested that nematodes can perceive chemical gradients while migrating in PF-127 gel matrix which mimics the three-dimensional perception and migration that occurs in natural soil (Wang et al., 2009a). Using our assay plate we demonstrate that
In conclusion, the new in vitro chemotaxis assay designed in the present study has allowed the easy and time-effective screening of various test compounds (both volatile and non-volatile), in a way that yields accurate representations of nematode responses to test compounds in a very short time (40 min for setting up the chemical gradient and 20 min for nematode behavioral response). Our assay could be very useful for screening a number of bioactive compounds for nematode behavior analysis on a large-scale. The results obtained may become useful for predicting the long-term fate of negative or positive interactions of nematodes with various test chemicals in field conditions. The application of this in vitro assay can also be extended to study the response of PPNs to various nematicides or drugs. Overall, our study provides a promising new tool for future investigation on PPN chemotaxis.