Molecular basis of acaricide resistance in tetranychus truncatus ehara (prostigmata: tetranychidae) infesting vegetable crops

Abstract

Spider mites of the family Tetranychidae are considered as one of the most serious sucking pests of vegetable crops worldwide. Intensive use of conventional acaricides had lead to the development of resistance in many mite species around the globe. In view of this, several novel acaricides with unique chemical structure and mode of action were introduced and commercialized for mite management. In Kerala, mite management in vegetable crops solely depends on the use of novel acaricides. Of late, several farmers have raised concern over their poor efficacy against mite pests. Spider mites have the ability to develop resistance quickly on continuous exposure to a pesticide. In this context, the present study was undertaken to investigate the status, biochemical and molecular bases of acaricide resistance in Tetranychus truncatus Ehara, the predominant species of spider mite infesting vegetable crops of Kerala. Purposive surveys were conducted in the vegetable fields of Vellanikkara, Thrissur and spider mites were collected and reared in the laboratory by assigning accession numbers. Three accessions/strains viz., VkOk1 (okra), VkAm3 (amaranthus) and VkPm3 (pumpkin) which were identified as T. truncatus were used for the study. Susceptibility of the three field strains to three commonly used acaricides, viz., spiromesifen, fenazaquin and diafenthiuron was evaluated in the laboratory following leaf dip bioassay in comparison with a laboratory maintained susceptible strain (SS). Bioassay study revealed that the strain VkOk1 recorded highest LC50 value and has developed 8, 13 and 10 fold resistance to spiromesifen, fenazaquin and diafenthiuron, respectively. This was followed by VkAm3 which showed 7.0, 5.53 and 1.67 fold resistance, while VkPm3 recorded 1.35, 1.13 and 1.03 fold resistance. The activity of the detoxifying enzymes viz., cytochrome P450 and carboxylesterase, was significantly higher in VkOk1strain followed by VkAm3. The strains VkOk1, VkAm3 and VkPm3 showed 2.69, 1.24 and 1.09 fold enhanced activity of Cytochrome P450, respectively compared to SS, while carboxylesterase in VkOk1 and VkAm3 showed an increased activity by 2.59 and 1.18 fold. However, the strain VkPm3 recorded a decrease in activity of carboxyleasterase by 0.78 fold compared to the susceptible strain. DNA isolated from the two resistant strains (VkOk1 and VkAm3) and the susceptible strain (SS) was amplified with the help of gene specific primers for cytochrome P450 and carboxylesterase. The results of PCR for cytochrome P450 gene showed that there was no amplification in the case of SS, whereas there were distinct markers in the resistant strains, okra and amaranthus at 1300 bp size. However, PCR amplification showed distinct markers for carboxyl esterase in all the three strains. The strains VkOk1 and SS showed markers at 1500 bp and 1300bp size respectively, while VkAm3 strain showed both the markers. The sequence homology search by BLASTn analysis showed that the sequences of cytochrome P450 of T. truncatus has similarities with cytochrome P450 sequences from different species of spider mites with an identity match ranging from 81 to 97 per cent, while carboxylesterase sequences showed similarity with two mRNA sequences of carboxylesterase of T. urticae. Further, the translated sequences of cytochrome P450 and carboxylesterase aminoacids from T. truncatus when analysed by BLAST P showed similarity with the amino acid sequences from other spider mites. The sequences of carboxylesterase genes from the resistant and susceptible strains did not align together, showing that there are two different caboxylesterase genes controlling the resistance to acaricides in T. truncatus. The study recorded resistance in T. truncatus to three novel acaricide molecules, for the first time from India. This is the first report of cytochrome P450 and carboxylesterase genes conferring resistance to acaricides in T. truncatus. The study also developed standard markers for discriminating the resistant and susceptible population in T. truncatus.