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

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Subject: search.filters.subject.Carboxylesterase

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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.
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    Elucidating the biochemical basis of interaction between tea mosquito bug, helopeltis antonii signoret (hemiptera:miridae) and cashew (anacardium occidentale)
    (Department of Agricultural Entomology, College of Horticulture, Vellanikkara, 2018) Nimisha, T; Deepthy, K B
    Cashew is one of the important foreign exchange earning crops of India with an export value of Rs. 5,077 crores during 2016-17.Cashew production is not in tune with increase in area under its cultivation. Low productivity in cashew is mainly due to the incidence of pests and diseases. Among the insect pests, tea mosquito bug (TMB), Helopeltis antonii Signoret (Hemiptera: Miridae) is the most important. It causes 30-50 per cent yield loss and during outbreak situations even up to 100 per cent loss has been reported. Cashew varieties exhibit wide variation in response to TMB infestation. Hardly any variety has ever been recorded as resistant to TMB till date. However, a few accessions are reported to be capable of withstanding TMB infestation and hence have been grouped as less susceptible category. Understanding the basis of interaction between the bug and the cashew is a prerequisite in developing varieties resistant to TMB. The present study entitled “Elucidating the biochemical basis of interaction between tea mosquito bug, Helopeltis antonii Signoret (Hemiptera: Miridae) and cashew (Anacardium occidentale)” was undertaken at the Department of Agricultural Entomology, College of Horticulture, Vellanikkara during September 2017 to July 2018. The objective of the study was to understand the variation in secondary metabolites and defense enzymes in selected cashew varieties induced by tea mosquito bug infestation and to elucidate the secondary metabolite detoxification mechanisms in the pest. Three months old grafts of four cashew varieties viz., two from the highly susceptible category (Anagha, Madakkathara-1) and two from less susceptible category (Damodar, Raghav) were used to conduct the experiment. Female adult bug (0-24 h old) was allowed to feed on each variety for different time intervals of 6, 24, 48 and 72 h. The biochemical parameters such as protein, phenol, tannin and defense enzymes viz., polyphenol oxidase (PPO) and phenyl alanine ammonia lyase (PAL) in leaves of TMB infested and non infested cashew grafts were analysed before release (0 h) as well as at different intervals of release. Biochemical analysis of the released TMB (crude homogenate of whole insect) viz., variation in total protein, SDS PAGE profiling of TMB crude protein, detoxifying enzymes viz., carboxyl esterase, cytochrome P450, and glutathione-s-transferases (GST) were carried out before releasing on the plant and at different intervals after release. The total leaf protein was found to be highest in the less susceptible variety Damodar (0.9925 mg g-1) and the lowest in the highly susceptible Madakkathara-1 (0.6729 mg g-1). Total phenol content was highest in the less susceptible Damodar (69.834 mg g-1) and Raghav (67.207 mg g-1) and the lowest was recorded in Anagha (29.625 mg g-1). Regardless of the varieties, highest phenol content was recorded in samples taken after 72 h of TMB infestation. Tannin content was also high in the less susceptible varieties Raghav (4.420 mg g-1) and Damodar (4.276 mg g-1) while highly susceptible varieties Anagha and Madakkathara-1 recorded lower values. Irrespective of the varieties, the highest tannin content was recorded in samples before the release of TMB (6.662 mg g-1). Results of the present study revealed that, detoxifying enzyme specific activity of PPO was highest in Damodar (0.003158 EU g-1min-1) and observed lowest value in Anagha (0.001406 EU g-1min-1). Irrespective of the varieties, enzyme activity reached highest at 48 h of release (0.00367 EU g1min-1). PAL specific activity was highest in Madakkathara-1 (0.575 μg g-1 min-1) and lowest in Anagha and was on par with Raghav and Damodar. In SDS PAGE, the whole body homogenate of adult female TMB revealed presence of bands in the range of ̴ 63 kDa and 35-48 kDa in the early stage of infestation. However, as the exposure time increased, protein bands ranging from 17-75 kDa appeared. Defensive enzymes viz., carboxyl esterase and GST expressed elevated activity in TMB that fed on less susceptible Raghav (258.117 μmol min-1mg-1 protein, and 365.262 μmol min-1mg-1 protein respectively) and Damodar (208.916 μmol min-1mg-1protein, and 501.879 μmol min-1mg-1protein respectively) when compared to the highly susceptible varieties. Cytochrome P450 showed highest activity in TMB fed on Damodar (0.372 nmol min-1mg-1 protein) and it was having lowest activity in insect fed on other three varieties. The study revealed that, the secondary metabolites viz., phenol, tannin and PPO have a definite role in imparting resistance in cashew to TMB attack. These metabolites have potential use in early detection of resistance in cashew against TMB. The enhanced levels of detoxification enzymes in TMB indicate plasticity of the pest against host plant defense and chance of resistance development against synthetic insecticides.