Fig 1.
Scheme of the nematode behavior tests and plant infection test.
(A) Dictyostelium discoideum cells or cell extract, cell-released material was placed in the yellow circle. Five to ten nematodes were placed 1.9 cm away from cells of D. discoideum and 1.2 cm away from the filter paper in the tester plate with phytagel. The deposit position of nematodes was indicated as “+”. The photograph of the tester plate was taken after 24 h and the trace of nematode movement was analyzed using ImageJ software. Trace in Region 1 indicates attraction and trace in Region 2 indicates repulsion. The trace of nematodes in each area was converted into the number of pixels, and the behavior was quantified. (B) Three L. japonicas seedlings (I-III) were placed separately in the square plastic plate with Lotus B&D medium. Dictyostelium cell extract was spotted and dried on semicircular filter paper. The filter paper was placed 2 mm from the side on the root tip.
Fig 2.
Differential responses of Meloidogyne incognita to Dictyostelium discoideum.
(A) Fruiting bodies (48 h), but not vegetative cells (0 h), of D. discoideum repels M. incognita. Values are expressed as mean±s.e. of N ≥ 13, ***P < 0.001 versus control (Student’s t-test, unpaired, two-tailed). (B)This repulsion activity remained in the tester plate gel after the removal of fruiting bodies. Values are expressed as mean±s.e. of N ≥ 13, ***P < 0.001 versus control (Student’s t-test, unpaired, two-tailed). 1 x 108 cells were used for each experiment.
Fig 3.
Responses of M. incognita to D. discoideum cell released materials and fruiting bodies extract.
(A) 74% of total trace was found in Region 2, indicating D. discoideum cell-released materials repelled M. incognita. Values are expressed as mean±s.e. of N ≥ 10, **P < 0.01 versus control (Student’s t-test, unpaired, two-tailed). (B) The cell extract showed repulsion activity in a dose-dependent manner. Values are expressed as mean±s.e. of N ≥ 17. *P < 0.05 and ***P < 0.001 versus control (One-way ANOVA followed by Tukey-Kramer test) (C)The cell extract from axenically grown D. discoideum also repelled M. incognita. Values are expressed as mean±s.e. of N ≥ 16. ***P < 0.001 versus control (Wilcoxon rank sum test).
Fig 4.
Dictyostelum species in different phylogenic group also repelled M. incognita.
In this experiment, each Dictyostelia species was cultured with E. coli on LP agar medium. D. purpureum (group 4), P. pallidum (group 2) and D. fasciculatum (group 1) cell extracts showed the same repulsion activity with that of D. discoideum (group 4). Values are expressed as mean±s.e. of N ≥ 16. ***P < 0.001 versus control (Kruskal-Wallis one-way analysis of variance followed by Dunnett test).
Fig 5.
D. discoideum cell extract protects plants from M. incognita infection.
(A) The number of infected RKNs of each seedling in plant infection test. Open bar indicates plant (I). Hatched bar indicates plant (II), and dotted bar indicates plant (III). Values are expressed as mean±s.e. of N ≥ 16. Bars with different letters denote a significant difference at P < 0.05 (Kruskal-Wallis one-way analysis of variance followed by Dunnett test). (B) The representative example of fuchsin staining of each plant. Bar indicates 500 μm.