Determination of preferred pH for root-knot nematode aggregation using pluronic F-127 gel

Abstract

Root-knot nematodes (Meloidogyne spp.) are obligate endoparasites of a wide range of plant species. The infective stage is attracted strongly to and enters host roots at the zone of elongation, but the compounds responsible for this attraction have not been identified. We developed a simple assay to investigate nematode response to chemical gradients that uses Pluronic F-127, a synthetic block copolymer that, as a 23% aqueous solution, forms a liquid at low temperature and a gel at room temperature. Test chemicals are put into a modified pipette tip, or 'chemical dispenser,' and dispensers are inserted into the gel in which nematodes have been dispersed. Meloidogyne hapla is attracted to pH gradients formed by acetic acid and several other Brønsted acids and aggregates between pH 4.5 and 5.4. While this pH range was attractive to all tested root-knot nematode strains and species, the level of aggregation depended on the species/strain assessed. For actively growing roots, the pH at the root surface is most acidic at the zone of elongation. This observation is consistent with the idea that low pH is an attractant for nematodes. Root-knot nematodes have been reported to be attracted to carbon dioxide, but our experiments suggest that the observed attraction may be due to acidification of solutions by dissolved CO(2) rather than to CO(2) itself. These results suggest that Pluronic F-127 gel will be broadly applicable for examining responses of a range of organisms to chemical gradients or to each other.

Fig. 1

Apparatus for CO₂ delivery to PF-127 gel. The lid of a standard Petri dish was modified with entry and exit ports for CO₂, and an entry port for air. Air could escape under the edge of the lid. An acrylic cylinder cemented under the lid just touched gel surface to maintain an area of water-saturated CO₂ on the gel surface. The apparatus is shown in cross section (A) and top view (B). The stippled region in A represents the gel with the nematodes

Fig. 2

pH gradient formation in PF-127 gel. The pH indicator bromocresol purple (pH 5.2–6.8, yellow to purple) was included in the gel. The dispensers initially contained 0.17 M acetic acid, and plates are shown 4 and 24 h after the dispenser was inserted into the gel

Fig. 3

Migration of Meloidogyne hapla VW9 J2 in acetic acid gradients in PF-127 gel. Dispensers contain 0.17 M, 0.34 M, or 0.85 M acetic acid as indicated at the top of the three columns of images. Dissecting microscope images of the dispenser small end were recorded at 5 h (A) and 24 h (B) after inserting dispensers in the gel. Scale bar = 1 mm. Lower magnification edge-lighted photographs of the dispenser large end at 24 h is shown in C. The diameter of the large end of the dispenser is ~ 4 mm

Fig. 4

Distribution of VW9 J2 in acetic acid gradients. Dispensers containing acetic acid in 23% PF-127 gel were immersed in buffered PF-127 gel containing 0.30 J2 per mm³. After 5 h, the gel was photographed with edge lighting. Ninety degree arcs, scaled to 0.5 mm wide in the original gel, were superimposed on the enlarged photographic images with the origin of the arcs at the middle of the dispenser wide end. J2 within each arc were counted for the 0.34 M (●) and 1.7 M (○) acetic acid dispensers (four dispensers from three plates), and the data were transformed to J2 per mm³ of gel. The average values and standard deviations are presented against distance from the dispenser large end. The standard deviation expanded at small distances from the dispenser because of the small volumes corresponding to the short arcs. Data were fit to Gaussian curves by a least-squares method

Fig. 5

Response of Meloidogyne hapla VW9 J2 to acid gradients in PF-127 gel. Each assay was in a Petri dish containing 6000 J2 in 20 ml of gel in 10 mM Tris-MES, pH7. Dispensers contained 0.40 M solutions of HCl (A), propionic acid (B, G), methane sulfonic acid (C), or pyridinium methanesulfonic acid (E), or 0.16 M citric acid (D, H), or water (F,I). All photographs were taken at 20 h after insertion of the dispensers. Panels A-F show the large end of the dispenser and were taken with edge lighting against a black background. Panels G-I show small opening of dispenser, and photomicrographs were taken under a dissecting microscope

Fig. 6

Scoring aggregation of root-knot nematodes to acetic acid. The small opening of the dispenser with 170 mM acetic acid is shown at 24 h after initiation of the assay. A. M. javanica strain VW4. This sample would be scored as “+” in Table 1. B. M. hapla strain LM. This sample was scored as “++.” C. M. hapla VW9 was scored as “+++.” Scale bar = 1 mm

Fig. 7

Response of Meloidogyne hapla VW9 to CO₂ in PF-127 gel. A. At 16 h after continuous exposure to CO₂, nematodes aggregated just outside the area exposed to CO₂ with pluronic gel buffered with 10 mM Tris-MES, pH 7. B. No aggregation was observed when gel was buffered with sodium acetate, pH 4.9. At the start of the assay, 9000 J2 were dispersed in 30 ml PF-127 and poured into the Petri dish. The images show an arc of the 1.5-cm diameter circle that was exposed to CO₂. Note that the number of J2 inside the circle is similar in A and B