PhD Thesis

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Subject: search.filters.subject.Leucinodes orbonalis

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    Mechanisms of resistance in Brinjal Shoot and fruit broer, Leucinodes orbonalis (Guenee) (Lepidoptera:Crambidae) to diamide insecticides
    (Department of Agricultural Entomology, College of Agriculture ,Vellanikkara, 2024-10-18) Anu Thomas
    The development of insecticide resistance among insect pests is a major concern in pest management. Generating data on the baseline susceptibility of field populations to insecticides facilitates to track the resistance development in insects. This helps in designing suitable insecticide resistance management (IRM) strategies; thereby, to delay the development of resistance and extend the useful life of an insecticide.Understanding the mechanisms of resistance, the possibilities of developing cross- and multiple-resistance, and the fitness costs involved in resistance are also crucial to maintain the sustainability of an insecticide. In this context, the present investigation entitled “Mechanisms of resistance in brinjal shoot and fruit borer, Leucinodes orbonalis (Guenee) (Lepidoptera: Crambidae) to diamide insecticides” was undertaken during 2019-2024 with the objectives to study the baseline susceptibility of brinjal shoot and fruit borer to diamide insecticides, to assess the possible mechanisms of resistance development, to investigate the potential of developing cross and multiple resistance in diamide-resistant populations and to analyze the fitness costs associated with resistance. Field populations of brinjal shoot and fruit borer were collected from brinjal fields of Kerala, Kullarayanpalayam (Palakkad) and Anchal (Kollam); Tamil Nadu, Devarayapuram (Coimbatore) and Trichy (Tiruchirappalli); and Karnataka, Heggadadevankote (Mysore) and Doddaballapur (Banglore Rural). A laboratory susceptible population, maintained without any insecticide exposure since 2012, was procured from NBAIR, Bangalore. The collected populations were assigned unique accession codes such as PKD, KLM, CMB, TRY, MYS, BAN, and Lo-S, respectively, and maintained separately in the laboratory. Information on insecticide usage pattern in brinjal cultivation obtained from farmers at collecting sites revealed the usage of insecticides of different modes of action and application above recommended dosages. An intensive application of various insecticdes including diamides was practiced in Bangalore Rural, Tiruchirappalli, Coimbatore, and Palakkad. In Kollam, on the other hand, a need-based application in recommended doses was followed. In Mysore, the farmers did not use any diamide insecticide; instead, they relied on organophosphates and neonicotinoids applied at frequent intervals. The laboratory bioassay of field populations of L. orbonalis was performed against flubendiamide (Fame 39.35% SC) and chlorantraniliprole (Coragen18.5% SC) to determine the median lethal concentration (LC50). The resistance ratio (RR) was assessed by comparing the LC50 values with that of the Lo-S population. All field populations of L. orbonalis except MYS displayed a significant shift in LC50 value compared to the Lo-S population and were considered resistant to flubendiamide and chlorantraniliprole based on the hypothesis of equality. Among the resistant populations, the BAN and TRY were resistant homozygotes according to the hypothesis of parallelism. The LC50 value of field populations ranged from 0.83 ppm in MYS to 544.07 ppm in TRY for flubendiamide compared to 0.50 ppm recorded with the Lo-S population. The TRY population showed the highest resistance ratio (RR) of 1079.5- fold to flubendiamide, followed by the BAN (845.85-fold), CMB (532.56-fold), and PKD (218.84-fold) populations. However, the KLM population showed comparatively less RR with 47.59-fold resistance, while the MYS population was considered susceptible to flubendiamide. The LC50 value for chlorantraniliprole ranged from 0.169 ppm in MYS to 116.80 ppm in BAN in contrast to 0.119 ppm recorded with the Lo-S population. Concerning chlorantraniliprole, the highest RR of 979.22-fold was recorded with the BAN population followed by 788.18-fold in TRY, 480.30-fold in the CMB, 244.75-fold in PKD, and 18.22-fold in KLM populations. The lowest RR of 1.41-fold was observed with MYS population and was considered susceptible to chlorantraniliprole based on the hypothesis of equality. The activity of detoxifying enzymes in the field populations of L. orbonalis was quantified and compared with the Lo-S population to examine the role of detoxifying enzymes in diamide resistance. The field populations, BAN, PKD, CMB, TRY, MYS, and KLM showed 1.66-, 1.47-, 1.24-, 1.21-, 1.21-, and 1.17-fold increase in carboxylesterase (CarE) activity, respectively. Significantly higher titers of cytochrome P450 (Cyt P450) activity were displayed by all the field populations with a 5.54- fold increase in the TRY population, followed by BAN (5.38-fold), CMB (5.26- fold), PKD (5.13-fold), MYS (4.56-fold), and KLM (3.12-fold). The relative activity of glutathione S-transferase (GST) was >4-fold in PKD, CMB, TRY, and BAN populations displaying 4.31, 4.33, 4.34, and 4.44-fold variations, respectively, compared to the Lo-S population. Meanwhile, KLM and MYS populations exhibited only 1.5-fold and 1.25-fold increases, respectively. To assess the potential for developing cross-resistance, a laboratory bioassay was carried out against cyantraniliprole, a less commonly used diamide insecticide. The field populations displaying resistance to flubendiamide and chlorantraniliprole also exhibited resistance to cyantraniliprole, even though the population had no previous exposure to the chemical. However, the populations that were not previously exposed to emamectin benzoate and spinosad did not show resistance to these insecticides, and thereby the possibility of developing multiple resistance in diamide-resistant populations was rejected. Fitness costs associated with diamide resistance were ascertained by comparing the life table parameters of different field populations of L. orbonalis with the Lo-S population. In the study, diamide-resistant populations, TRY, BAN, PKD, and CMB displayed significantly longer larval and pupal duration, and less oviposition period and female adult longevity than the Lo-S population. Male adult longevity in the TRY and BAN populations was also notably shortened compared to the Lo-S population. The survival of egg, larva, and pupa as well as fecundity of the diamide-resistant populations except KLM were significantly less compared to the Lo-S population. The BAN and TRY populations exhibited a relative fitness of 0.62, suggesting a higher fitness cost associated with diamide resistance. The CMB population displayed a relative fitness of 0.68 followed by PKD (0.74) and the lowest was in KLM (0.82). These results indicated a survival disadvantage with the resistant populations compared to the Lo-S population. In investigating the molecular basis of resistance in L. orbonalis, a partial sequence of the ryanodine receptor (RyR) gene involved in diamide resistance was analyzed. Thymine (T) was found to be substituted with cytosine (C) in the sequences of the BAN and TRY samples that were tested, but the Lo-S sample did not exhibit this substitution, which is consistent with the reference sequence (Acc. No. PQWD01009585.1) of L. orbonalis. Alignment of the protein sequences revealed a non-synonymous amino acid alteration from isoleucine to methionine (I to M) in the RyR of BAN and TRY, which was not observed in the Lo-S gene sequence, indicating the crucial role of point mutation for higher diamide resistance in TRY and BAN populations. The study revealed a shift in susceptibility status and the development of higher folds of resistance to diamide insecticides among the field populations of L. orbonalis. The possible role of detoxifying enzymes and target site mutations in the development of diamide resistance was also established. The study on cross- and multiple-resistance provided valuable information for the selection of insecticides in IRM programs. The involvement of fitness costs in the diamide-resistant populations indicated a negative impact of resistance on pest fitness and the chance of resistance reversion in the absence of insecticides.
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    Stability analysis and molecular characterization of F1 hybrids in brinjal (Solanum melongena L.)
    (Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, 2018) Kavishetti Vinay, Vishwanath; Lekha Rani, C
    Stability analysis helps in assessing genotype × environment interaction in order to identify stable genotypes in large multi-environment trials. Therefore the present study entitled “Stability analysis and molecular characterization of F1 hybrids in brinjal (Solanum melongena L.)” was carried out to evaluate ten hybrids along with one check across four locations. The locations selected for trials were College of Agriculture, Vellayani and farmer’s fields at Thiruvalla, Sadanandapuram and Kayamkulam in Kerala. The trial seasons were kharif (2015- 16) and summer (2016-17). The objective was to study the performance of superior hybrids over different locations and seasons from heterotic crosses of brinjal and to confirm the hybridity using SSR markers. Randomized block design with four replications was employed. Stability and adaptability of yield and yield attributing characters of hybrids were analysed by Eberhart-Russell model (1966). Pooled analysis of variance revealed significant differences among the genotypes, environments and genotype × environment interaction for all the characters studied. The indication was that the hybrids responded differently to changes in the environment. Promising hybrids were identified on the basis of stability parameters viz., overall mean, regression coefficient (bi) and deviation from regression (S2di). In kharif season, the hybrid Wardha local × Palakurthi local was observed to be stable and widely adapted to all environments for days to first flowering, number of fruits plant-1, fruit weight, fruit length, fruit girth, calyx length, yield plant-1, yield plot-1 and plant height. The hybrid Wardha local × Swetha was identified as stable with regard to fruit weight, yield plant-1 and yield plot-1. The hybrid Wardha local × Vellayani local was identified as stable for favourable environments with regard to fruit length, yield plant-1 and yield plot-1. The hybrid Surya × Vellayani local was found stable for poor environments with regard to days to first flowering. Stability analysis for the summer season crop also revealed the hybrid Wardha local × Palakurthi local as stable across all environments with respect to days to first flowering, days to first harvest, number of fruits plant-1, fruit weight, fruit length and yield plant-1. Swetha × Vellayani local hybrid was stable across all environments with regard to days to first harvest, fruit weight, fruit length, calyx length, yield plant-1, yield plot-1 and plant height. The hybrid Surya × NBR-38 was found stable with respect to yield plant-1, yield plot-1 and plant height for favourable environments. Qualitative characters viz., total phenols, total sugars and vitamin C content and biotic stress traits viz., shoot and fruit borer infestation and bacterial wilt incidence were recorded for the eleven hybrids in kharif and summer season in all four locations. On the basis of overall mean performance of hybrids, results illustrated that the hybrid Swetha × Vellayani local recorded the highest values for total phenols and the hybrid NBR-38 × Vellayani local recorded the lowest values. Total sugar content was highest in Wardha local × Swetha and NBR-38 × Selection Pooja and lowest in Surya × NBR-38. The hybrid Neelima and Swetha × Vellayani local recorded higher quantity of vitamin C and NBR-38 × Vellayani local and NBR-38 × Selection Pooja recorded the lowest values. The hybrids Wardha local × Palakurthi local, Swetha × Vellayani local and Neelima recorded minimum infestation of shoot and fruit borer and was found resistant to bacterial wilt also. Hybrids and their parental lines were characterized using SSR markers. Among the four markers studied, three markers viz., emb01M15, eme08D09 and CSM31 were found to be polymorphic among the parental lines of respective hybrids viz., Surya × NBR-38, Surya × Vellayani local, NBR-38 × Selection Pooja, Swetha × Vellayani local, Wardha local × Swetha, Wardha local × Palakurthi local, Palakurthi local × Vellayani local and NBR-38 × Vellayani local which could be used for ensuring the genetic purity of respective parental lines and hybrids. F2 families viz., Wardha local × Palakurthi local, Wardha local × Swetha, Wardha local × Vellayani local and Swetha × Vellayani local were selected on the basis of yield performance from F1 for further evaluation. F2 populations revealed that family Wardha local × Palakurthi local and Wardha local × Vellayani local were superior in yield performance and yield attributing characters. The present investigation revealed that the hybrids Wardha local × Palakurthi local and Swetha × Vellayani local were stable and widely adapted over different locations and seasons and the hybridity was confirmed with the SSR markers, emb01M15 and eme08D09.