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Fig. 1
Figure 1: Development of a novel PPN motility assay. (A, B) Outline of an assay plate. A center of agar medium is uplifted to form a volcano-shaped, round deck (ø 1 cm). (B) Exemplary setup of a motility assay. PPN (e.g., R. reniformis, white arrows) suspended in water are placed around a slope of a volcano mountain, and carefully spread up to an edge of deck (white line), while a testing reagent (e.g., cotton root extracts, grey arrow) dissolved also in water is loaded on a volcano deck and spread to a top side of edge (white dash line). Subsequently, the reaction and movement of PPN are observed by a stereomicroscope and photographed. In (A to C), a shape of the volcano mountain is outlined by solid black line.
Fig. 2
Figure 2: Validation of a novel PPN motility assay; R. reniformis is attracted toward cotton root extracts, but does not respond to peanut root extracts. (A) The time-resolved responses of R. reniformis upon the exposure to water, and root extracts prepared from 2-wk grown cotton plants (LONREN-1) and 3-wk old commercial peanut variety (Georgia-09B). Representative photographs are taken at 0, 12 and 18 hr of assays. Close up pictures of the boxed sections in 18 hr were shown in the right panel. Black lines draw the shapes of the volcano mountain, and white arrows indicate R. reniformis on the volcano deck. (B) Chemotactic behaviors of R. reniformis toward the root exudates of cotton and peanut plants. Numbers of R. reniformis relocated onto the volcano deck were counted at 18 hr post co-incubation with water and root extracts prepared from 2-wk grown cotton plants (LONREN-1) and 3-wk old commercial peanut varieties (Georgia-09B, FloRun107 and Tif Guard) (one-way ANOVA, N = 5). Asterisks (*) indicate statistically significant differences of chemotactic behaviors of R. reniformis toward the selected root extracts in comparison to water control by Dunnett’s P < 0.05.
Fig. 3
Figure 3: Root exudates of cotton plants attract R. reniformis. (A) Relocation of R. reniformis towards cotton root exudates. The time-resolved movement of R. reniformis toward cotton (LONREN-1) root exudates. Representative photographs are taken at 0 and 18 hr of assays. (B) Close up picture of a boxed section in (A at 18hr). White arrows indicate R. reniformis, and black arrows indicate water-insoluble precipitates of cotton root exudates on the deck. (C) Separation of hydrophilic supernatant (white arrow) and hydrophobic residue (black arrow) from total cotton root exudates (D, E). Responsive behaviors of R. reniformis toward hydrophilic (polar, D), and hydrophobic (nonpolar, E) compounds of cotton root exudates at 18 hr of assays. Polar, but not nonpolar, compounds of cotton exudates were able to attract R. reniformis. (F) Close up picture of a boxed area in (D), and white arrows indicate R. reniformis on the deck. In (E), black arrows indicate dark residues, partially water-undissolved nonpolar compounds of root exudates, which did not attract R. reniformis. In (A, D and E), white lines draw the shapes of the volcano mountain.
