Mutations in Acetylcholinesterase2 (ace2) increase the insensitivity of acetylcholinesterase to fosthiazate in the root-knot nematode Meloidogyne incognita

Abstract

The root-knot nematode Meloidogyne incognita causes severe damage to continuously cropping vegetables. The control of this nematode relies heavily on organophosphate nematicides in China. Here, we described resistance to the organophosphate nematicide fosthiazate in a greenhouse-collected resistant population (RP) and a laboratory susceptible population (SP) of M. incognita. Fosthiazate was 2.74-fold less toxic to nematodes from RP than that from SP. Quantitative real-time PCR revealed that the acetylcholinesterase2 (ace2) transcription level in the RP was significantly higher than that in the SP. Eighteen nonsynonymous amino acid differences in ace2 were observed between the cDNA fragments of the RP and SP. The acetylcholinesterase (AChE) protein activity in the RP was significantly reduced compared with that in the SP. After knocking down the ace2 gene, the ace2 transcription level was significantly decreased, but no negative impact on the infection of juveniles was observed. The 50% lethal concentration of the RNAi RP population decreased 40%, but the inhibition rate of fosthiazate against AChE activity was significantly increased in RP population. Thus, the increased fosthiazate insensitivity in the M. incognita resistant population was strongly associated with mutations in ace2. These results provide valuable insights into the resistance mechanism of root-knot nematode to organophosphate nematicides.

Figure 1. Alignment of the AChE2 amino acid sequences from susceptible (SP) and resistant (RP) populations of M. incognita.

The mutation sites are marked with asterisks. The catalytic triad residues, oxyanion hole and choline-binding site are respectively indicated with dots, solid triangles and quadrangles. The fragments used in RNAi are boxed. Amino acid sequences from ACE2 of Ditylenchus destructor and and Globodera pallida are used as homologous sequences.

Figure 2. Relative ace2 expression levels in susceptible (SP) and resistant (RP) populations of M. incognita.

(a) Transcription levels in RP and SP populations prior to ace2 gene interference using dsRNA. (b) Transcription levels in RP populations after the ace2 gene was subjected to interference using dsRNA. The transcription level of RNAi-egfp in the RP population was used as a control.

Figure 3. Insensitivity of AChE to fosthiazate in RP and SP populations of M. incognita.

(a) Insensitivity of AChE to fosthiazate in RP and SP populations prior to ace2 gene interference using dsRNA. (b) Insensitivity of AChE to fosthiazate in RP populations after ace2 gene interference using dsRNA. The inhibition rates of different fosthiazate concentrations against AChE were analysed using the minus logarithm of the concentration (−logC).