Abstract
Abstract Plant-parasitic, root-knot nematodes (Meloidogyne spp.) are a serious problem in agri- and horticultural crops worldwide. Understanding their complex host recognition process is essential for devising efficient and environmental-friendly management tactics. In this study, the authors report a new, simple, inexpensive, efficient, and quantitative method to analyze the chemotaxis of M. incognita second-stage juveniles (J2s) using a combination of pluronic gel and agar in a petri dish. The authors quantitatively defined the concentration gradient formation of acid fuchsin on the assay plate. Using this novel assay method, the authors have accurately measured the nematode response (attraction or repulsion) to various volatile (isoamyl alcohol, 1-butanol, benzaldehyde, 2-butanone, and 1-octanol) and non-volatile (root exudates of tomato, tobacco, and marigold) compounds. Isoamyl alcohol, 1-butanol, and 2-butanone were attractive to J2s through a broad range of concentrations. On the contrary, J2s were repelled when exposed to various concentrations of 1-octanol. Despite being attractive at lower concentrations, undiluted benzaldehyde was repulsive to J2s. Tomato and tobacco root exudates were attractive to J2s while marigold root exudates repelled J2s. The present quantitative assay method could be used as a reference to screen and identify new candidate molecules that attract or repel nematodes.
Highlights
Plant-parasitic nematodes (PPNs) especially rootknot (Meloidogyne spp.) and cysts cause substantial economic damage to vascular plants worldwide (Palomares-Rius et al, 2017)
Compared to the free-living worm, C. elegans, little is known about the olfactory behavior of PPNs (Rengarajan and Hallem, 2016) likely because of their obligate parasitic nature and lack of simple in vitro bioassays for quantitative analyses of PPN chemotaxis
We propose a simple, efficient, and reproducible new assay method to quantitatively analyze the chemotactic behavior of a PPN, i.e., M. incognita to a range of test compounds on combined agar and PF127 medium in a petri dish
Summary
A pure culture of M. incognita race 1 was multiplied on eggplant (cv. Pusa Purple Long) in a greenhouse. 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. 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). 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 Caenorhabditis elegans chemotaxis experiment on agar plate; Bargmann et al, 1993) were screened at six different concentrations (100, 10−1, 10−2, 10−3, 10−4, and 10−5). All individual treatments were statistically compared to negative controls, as stated in the figure legends
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