PhD Thesis

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    Abiotic stress tolerance in native isolates of Beauveria bassiana (Balsamo) Vuillemin (Hypocreales :Cordycipitaceae)
    (Department of Agricultural Entomology, College of Agriculture,Vellanikkara, 2024-07-22) Nimisha, T; KAU; Deepthy, K B
    Environmental factors such as high temperature, low humidity and soil acidity often limit the biological control potential of the entomopathogenic fungus (EPF), Beauveria bassiana. Identifying abiotic stress-tolerant B. bassiana isolates represents a possible strategy to overcome this problem. Native isolates of EPF tend to adapt more to the environmental stress conditions in the locality than the exotic isolates, which are exposed to a different microclimate and microbiota. Thus, this study aimed at identification of the abiotic stress tolerant native isolates of B. bassiana and elucidation of the biochemical and molecular mechanism of stress tolerance. Survey was conducted in ten districts of Kerala, across different agroclimatic zones (159 locations). Soil samples as well as field infected cadavers were collected during the survey. The physicochemical properties of the soil samples were analysed to understand prevailing abiotic stress conditions in the area of sample collection, from which the entomopathogenic fungi isolated. For soil isolation of EPF insect bait method and serial dilution and plating method were followed. Beauveria bassiana was not obtained from any of these soil samples. However, out of the 12 field infected cadavers collected, three were infected with B. bassiana. Beauveria bassiana was identified based on the morphological characters and later confirmed by molecular characterization. Sequencing of the ITS region (550 bp) revealed genetic differences among the isolates. The sequences were submitted to NCBI GenBank (National Center for Biotechnology Information) and the accession numbers were generated (BTL1 - OP271760, BTL2 - OP290199 and PKDE - OP292066). A maximum likelihood phylogenetic tree was built, and the evolutionary relationship among the isolates were also studied. Beauveria bassiana isolates (BTL1, BTL2 and PKDE) were grouped in a single cluster confirming their genetic relationship. Bioassay against third instar nymphs of cowpea aphid (Aphis craccivora Koch) revealed that at lower concentration of 105 spores/ml, only the PKDE isolate recorded cent percent mortality compared to other two native isolates as well as NBAIR (National Bureau of Agricultural Insect Resources) strain (Bb13). As the concentration of spore suspension increased to 107 spores/ml the PKDE and BTL2 isolates were on par with NBAIR strain in terms of LT50 values. The growth and biochemical parameters of the three native isolates of B. bassiana were studied under different abiotic stress conditions. The effects of temperature (28 - 40 oC), pH (2 - 6), salinity (0.5 - 2 M) and water stress induced by polyethylene glycol (PEG 10 - 45 %) on the growth of B. bassiana were assessed. Beauveria bassiana isolate PKDE (collected from Palakkad district) tolerated a temperature stress upto 40 oC. It also survived the extreme acidity (pH 2) and salinity (1.5 M) conditions. The B. bassiana isolate, PKDE was compatible with most of the commonly used insecticides viz., chlorantraniliprole, imidacloprid, thiamethoxam and spinosad. Among the fungicides tested, hexaconazole and carbendazim completely inhibited the growth of all the three isolates, while copper oxychloride showed 89 per cent compatibility with the isolate PKDE. The PKDE isolate of B. bassiana isolated from Palakkad district has shown exceptional resistance to the effects of temperature and drought stresses. Hence, biochemical characterization of this isolate was performed to confirm their ability of stress tolerance. Significant levels of trehalose content were recorded on exposure to heat (40 oC) and drought stress (45 % PEG) (20.33 mg/g of mycelia and 20.43 mg/g of mycelia, respectively) in the multiple stress tolerant PKDE isolate. A significant activity of catalase and peroxidase was also observed in response to heat stress at 40 oC in PKDE isolate (0.0072 EU/min/mg protein and 0.0602 EU/min/g tissue weight respectively), while activity was not significant with respect to drought. In PKDE isolte the mannitol dehydrogenase (MTD) and mannitol -1-phosphate dehydrogenase (MPD) displayed significantly increased activity upon exposure to temperature stress of 40 oC (0.363 and 0.317 EU/ min/ mg protein respectively) and drought stress (0.289 and 0.364 EU/min/ mg protein respectively) induced by 45% polyethylene glycol concentration compared to the control. Field studies concluded that two sprays of B. bassiana (PKDE, BTL2 and NBAIR strain) at a spore concentration of 1x108 spores/ml at 10 days interval, suppressed the cowpea aphid incidence. No mortality of natural enemies (coccinellid beetles and spiders) was observed in the treated plots. The protein profiling of PKDE isolate under stress conditions and without stress (control) was carried out to identify the molecular basis of stress tolerance. The results revealed that there was over-expression of proteins at high-temperature stress, and the molecular weight of proteins ranged between 11-17, 35-48, 48-63 and 100- 135 kDa. The relationship between heat shock proteins and thermotolerance in fungal biocontrol agents suggests a new approach for improving entomopathogenicity by enhancing the expression of thermotolerance-related proteins in conidia. This can be achieved by identifying fungal isolates with greater thermotolerance or by optimising the components of substrate for the growth of fungi to produce more thermotolerant conidia. The nucleotide sequence analysis in the neutral trehalase gene (Bb Nth1) and high osmolarity glycerol gene (Hog), which are known to be associated with multiple stress tolerance, revealed that Bb Nth1gene sequence of the three native isolates of B. bassiana contains a coding sequence (CDS) of 2232 bp which codes for 743 amino acids and the hog gene consists of 1077 bp which codes for 358 amino acids. The polymorphism analysis in the Bb Nth1 gene revealed that seven single nucleotide polymorphisms (SNPs) in the exon region and six SNPs in the intron region, in the BTL1 isolate. In the exon3 region of the BTL1 isolate one SNP was observed with G➔C transition and codon changed from GAG to GAC. The non-synonymous variation resulted in the substitution of glutamic acid to aspartic acid at 363rd position of amino acid sequence. In BTL2 isolate eight SNPs were found in the exon region and seven SNPs in the intron region. BTL2 isolate displayed two non-synonymous variations at the 363rd position (glutamic acid to aspartic acid substitution) and at 542th position (lysine to glutamic acid) of amino acid sequence. The second SNP observed in BTL2 isolate was with G➔A transition and codon changed from GAG to AAG. In the PKDE isolate, six SNPs were found in the exon region and none of which were non-synonymous variations. Only five SNPs were found in the intron region of PKDE isolate. Variant Effect Predictor software was used to determine the functional consequences of the observed SNPs. The two mutations observed in the Bb Nth1 gene resulted in a missense variant. The Protparam and HOPE software results also revealed that the mutation resulted in protein instability. While analysing the multiple sequence alignment of susceptible and multiple stress tolerant isolates, there was no non-synonymous variation in the Hog gene. The results of bioinformatics software such as Variant Effect Predictor and Protparam supported the above findings. The present study identified multiple stress tolerant isolate of B. bassiana (PKDE isolate) isolated from Palakkad district of Kerala which is safer to natural enemies and compatible with synthetic pesticides. This isolate may be successfully integrated as a microbial control agent in IPM programme.
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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.