1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)

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    Acaricide resistance in spider mites (Acari: Tetranychidae) Infesting major crops of Central Kerala
    (Agricultural Entomology, College of Agriculture, Vellanikkara,, 2025-08-18) Penuballi Swathi; Haseena Bhaskar
    Spider mites (Acari) inhabit a wide array of environments and pose significant threat to a variety of commercially grown vegetable crops and ornamental plants. Chemical management measures using synthetic acaricides remains the primary means of mite management, globally. However, the repeated use of chemical pesticides has led to the development of resistance in mite populations, including resistance to acaricides with novel modes of action, resulting in control failures. TetranychustruncatusEhara and Tetranychusgloveri Banks are the predominant spider mite pests on vegetable crops and ornamental plants in Kerala. Mite management in vegetable fields and horticultural nurseries of Kerala primarily relies on synthetic acaricides like spiromesifen, fenazaquin and diafenthiuron. Increased instances of control failures, despite the application of recommended doses of these acaricides, have recently been reported by the farmers and nursery owners. Sole dependence on acaricides for a prolonged period might have resulted in the reduced susceptibility of mite populations to commonly used acaricides. Hence, a study was carried out to investigate the susceptibility of the field populations of T. truncatus and T. gloveri to commonly used acaricides, the possible development of cross /multiple resistance and to elucidate the underlying mechanisms of resistance. Purposive sampling surveys were conducted across commercial horticultural nurseries, vegetable fields and tissue culture (TC) hardening units in central Kerala (Thrissur, Ernakulam, Palakkad and Malappuram) to collect spider mite populations. Isoline cultures of eight T. truncatus populations collected on marigold (Mg1Tr) and vegetable crops (Cp1Pt, Cu1Pt, Ok1Pt, Am2Pt, Am3Vt, Cp2Tv and Am4Tv), and nine T. gloveri populations collected on adenium (Ad1Rg and Ad2Sd), gerbera (Gr1Pt), dahlia (Da1Vt), anthurium (An1Bv) and tissue culture banana (Bn3Tr, Bn2Kn, Bn1Gn and Bn4Ak) were maintained in the laboratory with unique accession numbers. The laboratory maintained populations of T. truncatus (SST) for more than 10 years (>300 generations) and T. gloveri (SSO) for more than six years (>180 generations), without exposure to acaricides were designated as susceptible reference populations. The laboratory bioassay of T. truncatus with fenazaquin showed moderate levels of resistance in the populations on amaranthus (Am2Tr- 39.70 folds) and marigold (Mg1Tr- 25.60 folds) populations from Thrissur, cucumber from Pattambi (Cu1Pt- 12.90 folds) and amaranthus from Vyttila (Am3Vt- 10.50 folds). The other populations collected on amaranthus from Tavanur (Am4Tv) and on okra (Ok1Pt), and cowpea (Cp1Pt) from Pattambi, recorded low levels of resistance (RR 3.70- 7.90 folds). Among the T. gloveri populations, adenium population from National Rose Garden, Thrissur (Ad1Rg- 57.20 folds) and TC banana population from Kannara (Bn2Kn- 41.04 folds) recorded high levels of resistance to fenazaquin, while the mite populations collected on TC banana plants from Vyttila (Bn1Gn- 15.72 folds) and Thrissur (Bn3Tr- 15.80 folds), and on dahlia from Vyttila (Da1Vt- 13.31 folds) recorded moderate level of resistance. Other T. gloveri populations collected on gerbera, anthurium and adenium showed only low levels of resistance. Tetranychustruncatus collected on marigold (81.90 folds) and amaranthus (79.75 folds) from Thrissur recorded very high levels of spiromesifen resistance. The amaranthus population from Vyttila (Am3Vt- 33.41 folds); cowpea (Cp1Pt-25.16 folds) and okra (Ok1Pt- 25.83 folds) populations from Pattambi, showed moderate levels of resistance, while populations on cowpea (RR 07.08) and amaranthus (RR 05.90) collected from Tavanur recorded low levels of resistance. Susceptibility studies of T. gloveri with spiromesifen showed that the population on adenium from National Rose Garden (76.33 folds) and on TC banana from Kannara (71.33 folds) exhibited high levels of resistance. Populations collected on TC banana from Vyttila (30.33 folds), Thrissur (28.17 folds) and Anakkayam (13.79 folds) recorded moderate levels of resistance. The populations collected on the ornamental plants viz., dahlia (24.33 folds), anthurium (19.08 folds) and adenium (17.25 folds) also recorded moderate levels of resistance to spiromesifen. Low to moderate levels of resistance to diafenthiuron were recorded in T. truncatus (1.94- 17.56 folds) and T. gloveri (1.95- 15.57 folds), where marigold population from Thrissur (17.56 folds) and adenium population from National Rose Garden (15.57 folds) recorded significantly higher levels of resistance, respectively. Tetranychustruncatus and T. gloveri populations with resistance to fenpyroximate showed moderate levels of cross-resistance to fenazaquin and the propargite resistant populations showed cross resistance to diafenthiuron. However, the field populations recorded only low levels of multiple resistance to chlorfenapyr and hexythiazox (acaricides with different modes of action) in T. truncatus (1.20- 5.40 folds; 1.00- 1.50 folds) and T. gloveri (1.10- 2.35 folds; 2.08- 12.00 folds). Assay of detoxification enzymes in T. truncatus and T. gloveri showed significantly elevated activities of carboxylesterase (CarE) (1.27-2.27fold; 1.59-2.10 folds), cytochrome P450 monooxygenase (CytP450) (1.36- 4.98 folds; 1.08-3.10 folds), and glutathione S-transferase (GST) (1.00- 2.12 folds; 1.09- 1.98 folds) in resistant populations. The highest activity of detoxifying enzyme was recorded in T. truncatus population on marigold for CytP450. The study on differential gene expression and sequence variation of the CYP392E10 gene (encodes CytP450 enzyme) in the spiromesifen and fenazaquin resistant marigold population of T. truncatus showed a lower CT value with 7.61 folds higher gene expression than the susceptible population. Sequence analysis of CYP392E10 gene in the resistant population showed four single nucleotide polymorphisms (SNPs), where T is substituted by A (256th, 676th and 1356th) and A is replaced with G (708th position). The alignment of amino acid sequences of the susceptible and resistant T. truncatus populations showed substitution of Lleucine with methionine at 86th position and phenylalanine with isoleucine and lysine at 226th and 452nd positions, respectively. This study reports alarming levels of acaricide resistance in the spider mites, T. truncatus and T. gloveri in central Kerala, to commonly used acaricides. The mite species were also found to have developed significant levels of cross resistance to many unexposed acaricides. However, significantly low level of multiple resistance to the acaricides, chlorfenapyr and hexythiazox suggests their suitability as alternatives to commonly used acaricides in mite management. The study highlights the role of detoxifying enzymes and the overexpression of the related gene, particularly CytP450 in the development of resistance against commonly used acaricides (fenazaquin, spiromesifen and diafenthiuron). The study also provides new insights into the resistance mechanisms in the mite species in response to sustained acaricide pressure, exhibiting metabolic and target-site insensitivity, which further needs functional validation.
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    Molecular basis of acaricide resistance in tetranychus truncatus ehara (prostigmata: tetranychidae) infesting vegetable crops
    (Department of Plant Biotechnology, College of Horticulture, Vellanikkara, 2018) Anushree Bachhar; Haseena Bhaskar
    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.