Lauric acid in crown daisy root exudate potently regulates root-knot nematode chemotaxis and disrupts Mi-flp-18 expression to block infection

Abstract

Tomato (Solanum lycopersicum) crops can be severely damaged due to parasitism by the root-knot nematode (RKN) Meloidogyne incognita, but are protected when intercropped with crown daisy (Chrysanthemum coronarium L.). Root exudate may be the determining factor for this protection. An experiment using pots linked by a tube and Petri dish experiments were undertaken to confirm that tomato-crown daisy intercropping root exudate decreased the number of nematodes and alleviated nematode damage, and to determine crown daisy root exudate-regulated nematode chemotaxis. Following a gas chromatography-mass spectrometry assay, it was found that the intercropping protection was derived from the potent bioactivity of a specific root exudate component of crown daisy, namely lauric acid. The Mi-flp-18 gene, encoding an FMRFamide-like peptide neuromodulator, regulated nematode chemotaxis and infection by RNA interference. Moreover, it was shown that lauric acid acts as both a lethal trap and a repellent for M. incognita by specifically regulating Mi-flp-18 expression in a concentration-dependent manner. Low concentrations of lauric acid (0.5-2.0mM) attract M. incognita and consequently cause death, while high concentrations (4.0mM) repel M. incognita. This study elucidates how lauric acid in crown daisy root exudate regulates nematode chemotaxis and disrupts Mi-flp-18 expression to alleviate nematode damage, and presents a general methodology for studying signalling systems affected by plant root exudates in the rhizosphere. This could lead to the development of economical and feasible strategies for controlling plant-parasitic nematodes, and provide an alternative to the use of pesticides in farming systems.

Keywords: Chemotaxis; Meloidogyne incognita; Mi-flp-18; crown daisy; lauric acid; root exudate..

Fig. 1.

Root exudate reduced M. incognita numbers and suppressed nematode infection. Mono, monocropping; Inter, intercropping. (A) Diagram of the pot experiment linked by a tube, with the monocropping system in the left pot and the intercropping system in the right pot. (B, C) Root exudate reduced the number of nematodes in the tube and soils in the pot 35 d after inoculation. (D) Root exudate suppressed nematode infection by reducing root-knot numbers. The value of each bar represents the mean ±SE of n=4, where an asterisk denotes a significant difference at P < 0.05.

Fig. 2.

Identification and quantification of root exudate from tomato and crown daisy. (A) GC-MS chromatogram of tomato and crown daisy root exudate (black, green, and blue lines represent crown daisy, tomato, and the control, respectively). (B) Quantification of lauric acid in crown daisy root exudate. 1, lauric acid. The value of each bar represents the mean ±SE of n=3.

Fig. 3.

RNAi silencing of Mi-flp-18 in M. incognita J2. Soaking buffe, treatment with soaking buffer alone; gfp dsRNA, treatment with soaking buffer containing 1mg ml^–1 gfp dsRNA; flp-18 dsRNA, treatment with soaking buffer containing 1mg ml^–1 Mi-flp-18 dsRNA. (A) Fluorescence microscopy showing the ingestion of FITC in the soaking buffer by M. incognita J2s (scale bar, 10 μm). (B) Real-time PCR analysis of Mi-flp-18 transcript abundance. (C) Effects of Mi-flp-18 dsRNA on Mi-flp gene expression. The value of each bar represents the mean ±SE of n=3, where bars with different letters denote a significant difference at P < 0.05.

Fig. 4.

Mi-flp-18 is a pivotal gene regulating M. incognita chemotaxis and infection. Soaking buffer, treatment with soaking buffer alone; gfp dsRNA, treatment with soaking buffer containing 1mg ml^–1 gfp dsRNA; flp-18 dsRNA, treatment with soaking buffer containing 1mg ml^–1 Mi-flp-18 dsRNA. (A) Mi-flp-18 RNAi inhibited J2 chemotaxis in a Petri dish experiment. J2s immersed in three alternative treatments (soaking buffer alone, soaking buffer containing 1mg ml^–1 gfp dsRNA, or soaking buffer containing 1mg ml^–1 Mi-flp-18 dsRNA) were transferred to the Petri dish. (B, C) In the pot experiment, J2s inoculated with Mi-flp-18 dsRNA displayed inhibited infection ability (fewer root knot numbers). The value of each bar represents the mean ±SE of n=3, where bars with different letters denote a significant difference at P < 0.05.

Fig. 5.

Lauric acid affected M. incognita chemotaxis by regulating Mi-flp-18 expression and resulted in death. (A) Lauric acid concentrations of 0.5–4.0mM mediated J2 chemotaxis. (B) The death rate of J2s, caused by lauric acid. (C) Lauric acid regulated Mi-flp-18 expression in J2s. The value of each bar represents the mean ±SE of n=4, where bars with different letters denote a significant difference at P < 0.05.

Fig. 6.

The tomato–crown daisy intercropping system inhibited Mi-flp-18 expression in parasitism. The value of each bar represents the mean ±SE of n=4, where an asterisk denotes a significant difference at P < 0.05.

Fig. 7.

Schematic model demonstrating that root exudate in the tomato–crown daisy intercropping system may regulate J2 chemotaxis and infection by mediating Mi-flp-18 expression. LA, lauric acid.