PhD Thesis
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Item Baseline susceptibility of rice leaf folder Cnaphalocrocis medinalis (Guenee) (Lepidoptera: Crambidae) to selected insecticides(Department of Agricultural Entomology, College of Agriculture, Vellanikkara, 2025-08-29) Seena R Subhagan; Berin PathroseWith the rising food production demands, pesticides remain essential for achieving high yields, particularly in staple crops like rice. Despite the known environmental hazards of chemical pesticides, their repeated use has become common practice, often leading to selection pressure and the development of insecticide resistance. This issue is exacerbated when insect pests like the rice leaffolder, Cnaphalocrocis medinalis, which undergoes several generations a year, are exposed to the same insecticide across consecutive generations. Recent failures in controlling rice leaffolder outbreaks in Kerala have raised concerns, though no formal studies have documented insecticide resistance in these populations. This study aimed to address this gap by assessing the susceptibility of rice leaffolder populations to selected insecticides and investigating potential resistance mechanisms. Field populations from five agroecological units (AEUs) in Kerala were assessed for insecticide resistance through laboratory bioassays, with WYD (AEU 20) as a susceptible reference. The PKD (AEU 23), KUD (AEU 4), and TCR (AEU 6) populations experienced significant selection pressure, leading to moderate to high resistance to the tested insecticides, surpassing the resistance observed in the ONT (AEU 3) population. Resistance to acephate (5.07- to 172.53-fold) was identified for the first time in India. High resistance to quinalphos (133.24- to 611.37-fold) and carbosulfan (25.40- to 347.96-fold) were also noted. The observed carbosulfan resistance, despite no prior use, likely stems from cross-resistance due to previous organophosphate exposure, as both target acetylcholinesterase. Continuous exposure intensified resistance to lambda-cyhalothrin (up to 763.66-fold) and fipronil (up to 154.83-fold). Diamide resistance was significant, with chlorantraniliprole (1089.63 fold) and flubendiamide (1572.64-fold), marking the first reported flubendiamide resistance in C. medinalis in India. Cross-resistance to cartap (14.85- to 23.90-fold) and emamectin benzoate (24.97- to 81.09-fold) suggested resistance mechanisms driven by non-specific detoxification pathways. Further, the study aimed to elucidate the biochemical mechanisms of resistance by assessing the activities of detoxification enzymes. Resistant populations exhibited significantly elevated activities of carboxylesterase (CarE) (1.1–1.6-fold), cytochrome P450-dependent monooxygenase CytP450) (1.5–2.5-fold), and glutathione S transferase (GST) (2.3–3.0-fold). These findings underscore the increased activity of detoxifying enzymes as a contributing factor to the resistance observed in C. medinalis further confirmed through synergism bioassays. Bioassays with synergists revealed diverse resistance mechanisms across populations, driven by variations in detoxification enzyme activity. Metabolic resistance to acephate, quinalphos, carbosulfan, and lambda-cyhalothrin was primarily associated with CarE, CytP450, and GST, either individually or in combination. Multiple enzyme involvement was evident in PKD, KUD, and TCR, while CytP450 had a dominant role in ONT, highlighting enzyme-specific contributions to insecticide resistance. However, metabolic detoxification was not the primary driver of chlorantraniliprole and flubendiamide resistance in most populations, suggesting the involvement of alternative mechanisms. Minor contributions from CarE and CytP450 were detected in PKD, while slight synergistic effects in KUD may be linked to endosymbiont mediated resistance. In the case of fipronil, resistance was mediated by CytP450 in PKD, KUD, and ONT, whereas non-metabolic mechanisms likely contributed to the high fipronil resistance observed in TCR. These findings underscore the complexity of resistance mechanisms and highlight the need for further investigation into alternative pathways for diamide and fipronil resistance. This study investigated target site insensitivity as a potential resistance mechanism to diamides and fipronil in C. medinalis by analyzing mutations in the ryanodine receptor (RyR) and resistance to dieldrin (Rdl) genes. Gene duplication and a novel I4712N mutation in transmembrane (TM) domain 3 of RyR were detected in ONT and KUD populations, while PKD and TCR populations had a nonsynonymous mutation (F4691L) and a nonsense mutation (Y4692*) in the TM2-TM3 linker of the RyR gene, which may impact diamide binding. Additionally, a K4885K synonymous mutation was identified in TCR in the TM4-5 linker. As these mutations are newly reported, functional validation is required to confirm their role in resistance. Molecular analysis of the Rdl gene identified an A282S mutation in TM 2 of all populations, including the susceptible WYD, suggesting a limited role in fipronil resistance. Notably, this study reports a novel V457F mutation in TM 4 of the Rdl gene in the resistant TCR population, which may have contributed to high fipronil resistance (154.83-fold) by altering GABA receptor function. The absence of target-site mutations for isoxazoline and meta-diamides suggests their continued efficacy against C. medinalis. Metagenome analysis identified Pantoea sp. and Wolbachia sp. as potential endosymbiont contributors to microbial detoxification of chlorantraniliprole and flubendiamide resistance in KUD. These findings provide new insights into resistance mechanisms and highlight the need for further functional confirmation. Rice leaffolder populations in Kerala have developed diverse resistance mechanisms in response to sustained insecticide pressure, exhibiting metabolic and target-site adaptations. This study presents the first detailed evaluation of insecticide resistance in C. medinalis from Kerala, uncovering alarming resistance levels to multiple insecticides, including newly documented cases for acephate, carbosulfan, lambda-cyhalothrin, and flubendiamide. Metabolic detoxification, primarily through CarE, CytP450, and GST, played a key role in resistance against organophosphates, carbamates, and synthetic pyrethroids, with multiple detoxification pathways raising concerns about cross-resistance. Mutations in the RyR and Rdl genes suggest target site insensitivity for diamides and fipronil (TCR), while the potential involvement of endosymbionts in microbial detoxification (KUD) adds another layer of complexity. These findings highlight the urgent need for proactive resistance management strategies, integrating insecticide rotation, biological control, and molecular monitoring to sustain effective pest management in Kerala’s rice ecosystems.Item Nucleopolyhedrovirus mediated biointensive management of polyphagous pest Spodoptera litura F.(Department of Agricultural Entomology,College of Agriculture,Vellayani, 2025-09-12) Binseena,S R; Faizal,M HThe study entitled ‘Nucleopolyhedrovirus mediated biointensive management of polyphagous pest Spodoptera litura F. was carried out at the Department of Entomology, College of Agriculture, Vellayani, during 2019-2024 with the objective of development and evaluation of liquid and bait formulations of Spodoptera litura nucleopolyhedrovirus (SlNPV) incorporating botanicals and identification of viral gene sequences (iap and egt) of pesticidal relevance in SlNPV genome. Bioassay of botanical oils such as neem, pongam, and cashew nut shell liquid (CNSL) was done against S. litura under laboratory conditions. Cent percentage mortality of larvae was observed for neem oil 4 % and pongam oil 10 % treatments at 144 HAT and CNSL 5 % at 168 HAT. The LT50 values of the botanical oils were computed to 142.45, 146.37, and 176.54 hours for neem oil 4 %, pongam oil 10 %, and CNSL 3 % respectively with corresponding LT90 values of 329.04, 625.90, and 357.84 hours. In the bioassay of SlNPV, 100 % mortality was caused by the treatments with 1010 and 109 POB mL-1 which was at par with the mortality caused by lower concentrations of 108 and 107 POB mL-1 at 192 HAT. The effective doses (ED) of neem oil, pongam oil and CNSL were arrived at as 3.4 %, 4 %, and 2.5 % respectively by critically assessing the three important parameters of mortality, LC50 value and leaf area damage. A combination treatment of SlNPV (107 POB mL-1) and different doses (ED, ¾ ED, ½ ED, and ¼ ED) of botanical oils was attempted with the aim of reducing the lethal time of SlNPV. Significantly high mortality of S. litura (53.33 %) could be achieved in the combination treatments of SlNPV + neem oil 2.5 % as early as 48 HAT. However, at 144 HAT both SlNPV + neem oil 2.5 % and SlNPV + pongam oil 3 % treatments could result in 93.33 % mortality as against 60 % for treatment with SlNPV alone. The development of emulsifiable suspension (ES) formulations of SlNPV was attempted employing different oils as base. Isopropanol was identified as the solvent since it could solubilize all the oils tried at 5 % concentration, with low turbidity and viscosity. Emulsifier combinations of Span 20 + Triton X -100 (29:71) at 5 % for neem oil, sunflower oil, coconut oil, mustard oil, as well as Span 20 + Tween 80 (47:53) at 12 % for pongam oil, all yielding HLB value of 12 produced excellent bloom of Score 5 upon emulsification. SlNPV was dispensed into the oil-solvent-emulsifier combination so as to yield a final concentration of 1010 POBs mL-1. Among the five ES formulations evaluated, neem oil based (ES1) and pongam oil based (ES2) formulations were found to be superior producing significantly high mortality of 100 % and 93.33 % respectively even after 7 months of storage. Boric acid 1 % was selected and added to the formulation since it yielded superior protection of SlNPV with OAR (original activity remaining) value of 65.72 % upon exposure to sunlight. Development of storable ready to use bait formulations containing SlNPV and botanical oils were attempted, utilizing selected combination base matrices containing any two of wheat bran (WB), wheat flour (WF), rice bran (RB), and chickpea flour (CF) along with additives (A) (jaggery, cornstarch, CMC) as well as artificial diet alone. Based on the feeding preference and bait consumption by S. litura in free-choice and no-choice situations respectively three base matrices viz., artificial diet, WF (75 %) + CF (25 %) and WB (65 %) + WF (35 %) + A were selected, into which SlNPV (107 POB g-1) and either neem oil (2.5 %) or pongam oil (3 %) were infused. All three baits thus developed exhibited satisfactory structural integrity upon dehydration and rehydration. WB + WF based two bait formulations B3 and B2 of SlNPV containing pongam oil and neem oil respectively, were found to produce significantly high mortality of S. litura (100 % and 93.33 % respectively). Superior emulsifiable suspension and bait formulations alone and in combinations of the ones having the same botanical oil were evaluated for the management of S. litura in okra under polyhouse conditions. Combined application of an emulsion of ES2 and bait formulation B3 containing both SlNPV and pongam oil produced 96.67 % mortality of S. litura at 7 DAT, an effect on par with the chemical insecticide Flubendiamide 39.35 % SC. Identification of viral genes iap and egt, of pesticidal relevance was attempted in SlNPV for which specific primers with oligonucleotide sequences forward 5' GATTCGATCGCTGTCAACCT-3' reverse 5'-TTTCACTTTGGATGCTGCCT-3' and forward 5'-ATGGACTCGAACATGTTGGAC-3' reverse 5' AAGTCGAACATTGCGTATTTGG-3' respectively were constructed. Amplicons of size 500 bp and 600 bp could be detected in PCR with these primers, indicating the presence of both iap and egt sequences in SlNPV. The combined application of emulsifiable formulation ES2 [containing SlNPV (1010 POB mL-1), pongam oil (82 %), isopropyl alcohol (5 %), Span 20 + Tween 80 in the ratio 47:53 (12 %) and boric acid (1 %)] and bait formulation B3 [containing SlNPV (107 POB g-1 ), pongam oil (3 %), base matrix of WB + WF in the ratio 65:35 (77 %), jaggery (19 %), corn starch (0.3 %) and CMC (0.7 %)], developed in the present study each delivering SlNPV and pongam oil @ 107 POB mL-1/g-1 and 3 % respectively upon application was found effective in managing S. litura and have the potential to be developed as the predominant eco-friendly tactic in its IPM.Item Efficacy and biosafety evaluation of the entomopathogenic fungus Lecanicilliumsaksenae (Kushwaha) Kurihara and Sukarno(Department of Agricultural Entomology, College of Agriculture, Vellayani, 2025-07-17) Karthik, R.S; Reji Rani, O PThe investigation entitled “Efficacy and biosafety evaluation of the entomopathogenic fungus Lecanicilliumsaksenae (Kushwaha) Kurihara and Sukarno” was carried out during 2019 to 2024 at the Department of Entomology, College of Agriculture, Vellayani. The objectives of the study were to evaluate the efficacy of bioformulations of L. saksenae in managing the cowpea pod bug Riptortuspedestris, and to evaluate its biosafety to non-target organisms, including productive insects, pollinators, natural enemies, and mammals. A field experiment conducted in cowpea variety Jyothika, with five treatments viz. (T1) chitin enriched oil formulation of L. saksenae, 108 spores mL-1 @ 10 mL L-1 (T2) talc based formulation of L. saksenae, 108 spores mL -1 @ 20 g L-1 , (T3) L. saksenae spore suspension, 108 spores mL -1 @ 20 mL L-1, (T4) talc formulation of L. lecanii, 108 spores mL-1 @ 20 g L-1 (T5) dimethoate 30 % EC @ 1.5mL L-1. Of these, foliar application of chitin enriched oil formulation of L. saksenae was the superior treatment as it recorded a significantly lower population of the pod bug, R. pedestris. The reduction in population was 34 per cent after the first application and by 21.33 per cent after the second spray, which was equally effective as the insecticide dimethoate 30% EC sprayed at 1.5mL L-1. Population of predators such as the coccinellid beetle, Coccinellatransversalis and spiders Lycosapseudoannulata and Oxyopes sp. did not vary significantly, indicating its safety to natural enemies associated with cowpea ecosystem. Experiment to assess the safety of L. saksenae to non-target organisms was carried out using a concentration that is tenfold higher (109 spores mL-1) than the infective dose to insects. Safety to productive insects was examined using Indian bee, Apisceranaindica and the stingless bee Tetragonulairidipennis. L. saksenae conidial suspension exposed by dry film method did not cause any symptoms of mycosis such as irritability, sluggishness or disoriented movements in adult bees throughout the observation period. Topical application of the conidial suspension did not exhibit any significant difference in the colony strength, brood area (139 to 272.5 cm2) and storage area (31 to 136 cm2) of honey and pollen. There were no behavioural abnormalities nor symptoms of mycosis during the experimental period of ten weeks and the brood development was comparable with that of untreated colonies. The safety test carried out in adult T. iridipennis using the same method, revealed that the fungus was not pathogenic to it, as there were no symptoms of mycosis such as irritability, sluggishness or disoriented movements, till the fourth day post treatment. The colonies treated with conidial suspension did not exhibit symptoms of mycosis or mortality. The coccinellid predator, C. transversalis treated using dry film revealed no symptoms indicative of fungal infection. The cumulative mortality of treated grubs observed for a period of five weeks did not differ significantly with that of untreated (90 and 100 per cent respectively). They pupated normally and the percentage adult emergence was comparable with that of the untreated group (40 per cent each). The larval syrphid predator Dideopsisaegrota tested for its susceptibility to L. saksenae completed its life cycle normally with no symptoms of fungal infection. The mortality recorded four days after treatment was 40 per cent in the treated as well as control group. The larval parasitoidBraconbrevicornis, exposed to the conidial suspension by dry film method did not develop any disease. The cumulative mean mortality of adults did not differ significantly till 12 days of treatment between the treated and untreated (98-100 per cent). Similarly, there was no symptoms of infection in the larval parasitoidGoniozusnephantidis till seven weeks of exposure. The mortality recorded was on par (74 -76 per cent) in both the groups after six weeks of treatment. The egg parasitoidTrichogrammachilonis, emerged normally after treatment and the rate of adult emergence was 11.31 to 94.30 in treated and 14.76 to 98.46 in control, which did not vary significantly from each other. The pollinators Halictus sp. and D. aegrota. topically treated with L. saksenae showed no signs of mycosis, abnormal behavior, or mortality, further validating the safety of L. saksenae to beneficial insects. To evaluate biosafety of L. saksenae in mammals, the experiment was carried out in Department of Veterinary Pathology, College of Veterinary and Animal Sciences, Mannuthy. The acute toxicity studies were conducted on eight weeks old healthy Wistar albino rats (Rattusnorvegicus) weighing 150 – 200 g. The experiment was laid in CRD with three routes of administration viz. oral, dermal, intranasal administration of L. saksenae (6 × 109 spores mL⁻¹) and a control group with six animals per route of adminstraion. Treated animals were observed for clinical, haematological parameters and histopathological changes. The results revealed that there was no variation in body temperature, food and water intake and weight gain during the experimental period. Haematological parameters such as RBC, WBC, haemoglobin, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), granulocyte count, agranulocyte count, and platelet count were within the normal range in the treated and untreated animals. Serum biochemical analysis of orally treated animals performed to estimate liver function parameters, including alkaline phosphatase (ALP), serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), total protein, albumin, and globulin, as well as the renal function parameters, including urea, creatinine, and uric acid. There were no abnormalities in the test results obtained. Euthanized animals exhibited normal gross morphology and histopathological observations did not reveal any significant pathological lesions in the vital organs including brain, liver, kidneys, lungs, spleen, skin and intestines, confirming the non pathogenic nature of L. saksenae to mammals. In conclusion, the chitin enriched oil formulation of L. saksenae offers a viable alternative to chemical pesticides for managing the major sucking pest of cowpea, the pod bug, R. pedestris, without compromising the safety of natural enemies. The fungus also proved safe to productive insects, pollinators and mammals, strongly supporting its candidature in sustainable and ecofriendly pest management programmes.Item Endophytic and growth promoting activities of the entomopathogenic fungus, Lecanicilliumsaksenae (Kushwaha) Kurihara and Sukarno in rice(Department of Agricultural Entomology, College of Agriculture, Vellayani, 2025-07-11) Shree Naveena, PThe study entitled “Endophytic and growth promoting activities of the entomopathogenic fungus, Lecanicilliumsaksenae (Kushwaha) Kurihara and Sukarno in rice” was carried out during 2020-2024 in the Department of Agricultural Entomology, College of Agriculture, Vellayani, Thiruvananthapuram. The objective of the study was to establish the entomopathogenic, endophytic and growth promoting attributes of L. saksenae in rice. The infection process of L. saksenae in the rice bug, investigated using Scanning Electron Microscopy (SEM), revealed five step infection processes comprising adhesion, germination, penetration, colonisation/invasion, and conidiation/dissemination at 24, 48, 72, 96 and 120 hours post inoculation (HPI), culminating in dissemination of spores facilitating horizontal transmission and complete mycosis evidenced by mummified appearance, by 144 HPI. Biochemical mechanisms involved in pathogenesis were deciphered using metabolomic analyses of the infected rice bug. Gas Chromatography-Mass Spectrometry (GC-MS) and High-Resolution ORBITRAP Liquid Chromatography Mass Spectrometry (HR-LC-MS) revealed an array of 19 insecticidal compounds, three immunosuppressors, three antimicrobial compounds, and 13 other metabolites related to insect metabolism, highlighting the complex biochemical arsenal employed by L. saksenae in insect pathogenesis. In addition to entomopathogenicity, L. saksenae exhibited significant plant growth-promoting traits. Plate assays and spectrophotometric observations, displayed elevated levels of phytohormones, notably gibberellic acid (GA3: 334.68 µg mL-1) and indole acetic acid (IAA: 30.00 µg mL-1). It also exhibited zinc and phosphate solubilization efficiency (1.15 SE and 1.70 SE), which are related to nutrient assimilation. The siderophore (2.23 AU) and ammonia production (15.42 µmol mL-1) levels which help in nutrient uptake were significant and higher than those in other entomopathogenic fungi. The study further examined endophytic association of L. saksenae upon seed inoculation, in rice through re-isolation, microscopy, and PCR. The fungus could be reisolated from roots, stems, and leaves of the inoculated plants up to 90 days after seed inoculation (DAI) demonstrating its systemic translocation within then plant. Maximum colonisation was noted in roots (72.86 per cent), followed by stem (64.29 per cent) and leaves (40.00 per cent) at 30 DAI, which tapered to 32.86, 17.14 and 11.4 per cent at 90 DAI. SEM observations confirmed internal colonisation of L. saksenae in the leaf stem and root tissues of inoculated rice, which was further established by PCR amplification, that unequivocally matched with the original Accession. MN545844 deposited by Rani et al. (2015). Comparative metabolomic analysis between inoculated and uninoculated plants revealed significant biochemical changes due to L. saksenae colonisation. Significant elevation of proteins, sugars, and phenolic compounds in the inoculated plants indicated a metabolic shift that favours enhanced growth and defence. Untargeted GC-MS analysis revealed predominance of carbohydrates at maximum tillering stage which indicates protection at cellular levels and promotion of plant growth. Untargeted ORBITRAP HR LC-MS detected the presence of 10 insecticidal/insectistatic, one nematicidal, and three antimicrobial compounds, suggesting that L. saksenae induced the defence mechanism in inoculated plants. LC-MS/MS revealed profound modulation of growth and defence related hormones at critical plant growth stages due to fungal inoculation. Inoculated plants showed increased growth related hormones such as indole-3-butyric acid (IBA), IAA, GAs, N6-benzylaminopurine (6-BAP), trans-zeatin (tZ), and trans-zeatin riboside (tZR) at the seedling stage, and IAA, IBA, GA3, GA4, GA7, and tZR at the maximum tillering stage. Defence hormones such as abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), cis-jasmone (CJ), methyl jasmonate (MeJA), epibrassinolide (EBL), and 1 aminocyclopropane-1-carboxylic acid (ACC) were significantly higher in the inoculated plants at the seedling stage and SA, JA, CJ, MeJ, and ACC at the tillering stage, which are related to increased resistance to pest infestation. Inoculated seeds exhibited improved germination (6.38 per cent) and the seedlings exhibited increase in shoot and root length (30.30 per cent, 55.58 per cent), number of leaves and roots (35.29 per cent, 26.58 per cent) and the seedling vigor index (51.46 per cent), compared to the control. Pot culture experiments proved enhancedplant height (25.80 per cent), number of tillers (36.99 per cent), panicle count (30.00 per cent), grain number (24.20 per cent), grain yield (30.00 per cent), and straw yield (28.43 per cent) in the inoculated plants. In L. saksenae colonised, rice bug infested plants, there was a significant reduction in the number of eggs laid (29.40 per cent), number of feeding punctures (32.83 per cent) and grain damage (37.45 per cent) compared to control plants. Gene expression analysis revealed the upregulation of defence related genes, phenyl ammonia lyase (PAL) and lipoxygenase (LOX) in both inoculated and control plants, indicating it as a general stress response to herbivory. However, the pathogenicity related genes of the entomopathogens viz. subtilin like protease (PR1) and chitinase II (CHIT II) were not expressed in the colonised plants, as these genes are typically involved in breaching of insect cuticle. Headspace volatile analysis (GC-MS) of L. saksenae inoculated plants before rice bug infestation revealed the presence of 2-heptanone (natural enemy attractant and insect repellent), and 4-methylbenzaldehyde (oviposition deterrent). L. saksenae inoculated, rice bug infested plants, displayed defence related volatiles, such as S linalool (natural enemy attractant and insect repellent), methyl salicylate (natural enemy attractant), 9-octadecenamide (Z) (insecticidal), N-methyl-1-adamantaneacetamide (insecticidal and repellent). Additionally, there was upregulation of defence related enzymes, PAL, catalase (CAT), superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (POD), and ascorbate peroxide (APX) in inoculated plants. Temporal profiling revealed a dynamic activation pattern, where PPO, SOD, and APX peaked early at 48 hours after insect release (HAR), followed by a surge in PAL and POD at 72 HAR, and a delayed peak of CAT at 96 HAR. Importantly, L. saksenae inoculated plants displayed earlier and more intense upregulation of SOD and PAL, indicating a primed defence in plants during herbivore attack. These biochemical changes may underlie the observed reduction in pest performance and feeding efficiency. The above findings established the entomopathogenic, endophytic and growth promoting attributes of the indigenous isolate, L. saksenae in rice. By elucidating the tritrophic interactions involving the fungus, plant and insect, the study positions L. saksenae, as a "two-in-one" eco-friendly solution for plant health management in rice.Item Bio-efficacy of the entomo pathogenic bacteria, photorhavdus luminescens(Thomas & Poinar) aganist Tetranychus truncatus Ehara (Prostigmata: Tetranychidae).(Department of Agricultural Entomology, College of Agriculture, Vellanikkara, 2024-08-31) Athira, G Menon.; Haseena, BhaskarSpider mites pose a serious threat to economically important agricultural and horticultural crops, worldwide. The spider mite, Tetranychus truncatus Ehara is a widely distributed polyphagous species inflicting considerable damage to vegetable crops and ornamental plants, both under open and protected cultivation in Kerala. The repeated use of synthetic acaricides has led to the development of resistance in T. truncatus against novel acaricides such as fenazaquin and spiromesifen, necessitating more sustainable alternative strategies for mite pest management. The symbiotic bacterium, Photorhabdus luminescens associated with the entomopathogenic nematodes (EPNs), Heterorhabditis spp. has been reported as a potential biocontrol agent against a broad range of arthropod pests, including mites. The research programme on ‘Bio-efficacy of the entomopathogenic bacteria, Photorhabdus luminescens (Thomas & Poinar) against Tetranychus truncatus Ehara (Prostigmata: Tetranychidae)’ was carried out during 2018-2023 with the objectives to identify the potent local strains of P. luminescens; to develop suitable formulations of P. luminescens and to evaluate the efficacy of bio-formulations of P. luminescens against T. truncatus. Soil samples were collected from different localities across five districts of Kerala viz., Kasaragod, Malappuram, Thrissur, Ernakulam, and Idukki. In the laboratory, EPNs were isolated from 14 samples following bait trapping with Galleria mellonella larva, where the per cent infection ranged between 40 to 100 per cent. Bacteria were isolated from the EPN-infected cadavers of G. mellonella (14 isolates), as well as from the Heterorhabditis-infected Galleria cadavers received from CPCRI and NBAIR (2 isolates). Cultural characterization of the bacterial isolates showed circular to irregular, entire, opaque and smooth bacterial colonies on nutrient bromothymol blue agar (NBTA) medium. All the isolates showed negative reactions to the Gram staining and the bacterial cells were rod shaped. The bacterial colonies showed varying degrees of red colour from pinkish red to brick red in NBTA media. For molecular characterization, the 16S rRNA gene of the isolates was amplified, sequenced and subjected to BLASTn for homology search. One species of EPN symbiotic bacteria, Photorhabdus luminescens (2 isolates) and nine non-symbiotic/ associated bacteria viz., Stenotrophomonas maltophilia (2 isolates), Alcaligenes aquatilis (1 isolate), Brevundimonas diminuta (1 isolate), Brucella pseudointermedia (1 isolate),Ochrobactrum sp. (1 isolate), Brucella pseudogrignonensis (1 isolate), Brucella anthropi (1 isolate), Pseudomonas azatoformans (2 isolates) and Pseudomonas lactis (4 isolates) were identified. A phylogenetic tree was constructed based on the gene sequences of 16S rRNA, to validate the bacterial identity. The two isolates of P. luminescens (ODA and CNT1) obtained in the study were used for the bio-efficacy studies on eggs and adults of T. truncatus. The Cell Suspension (CS) and Cell-Free Supernatant (CFS) of both the isolates showed significant ovicidal and adulticidal effects against T. truncatus, after 72h of treatment application. In general, the mortality rate increased with an increase in the concentration of CS/CFS treatments. At the concentration of 108 cells/ml, the CFS and CS treatments of ODA isolate recorded egg mortality of 84.00 and 44.00 per cent, while CNT1 isolate recorded 58.67 and 84.00 per cent egg mortality, respectively. At the highest concentration, ODA isolate exhibited significantly higher mortality of adult mite for both CS (98.67%) and CFS (100%). Similarly, CNT1 isolate recorded adult mortality of 100 and 98.67 per cent, respectively for CS and CFS treatments. The quantitative assay of protein recorded 238.5 mg/ml and 148. 05 mg/ml of protein in the bacterial supernatant of ODA and CNT1 isolates, respectively. The qualitative analysis of protein was carried out using SDS PAGE. Three protein bands at 25-29 kDa and a single protein band at 42 kDa were obtained. Toxin proteins corresponding to these molecular weights were reported to possess significant insecticidal effects. The P. luminescens ODA isolate that recorded the highest mortality of T.truncatus for both CS and CFS treatments in the laboratory bioassays, as well as a higher growth rate on NBTA media was selected for the preparation of bio- formulations. The solid formulations using 1000g of starch and 3300g of talc per 1000 ml of culture media yielded perfect powder-like consistency, respectively for starch- based and talc-based formulation. Glycerol, trehalose, starch and polyvinyl propylene (PVP) were used as additives to prepare liquid formulations. Four liquid and two solid formulations developed in the study were evaluated against eggs and active stages of T. truncatus, separately in laboratory bioassays. All the formulations of P. luminescens (2.5×108 cells/ml) showed significant ovicidal activity at 72h of treatment application. The PVP-based liquid formulation recorded significantly higher mortality of eggs (97.33 %) and adults (100 %), followed by the starch-based solid formulation (82.67 and 90.67%, respectively). Study on the shelf life of the formulations showed that the bacterial population declined from the day of inoculation (DAI) to 90 DAI, both under refrigerated and room temperature storage conditions. In liquid formulation, the population declined from 3.6×108 cfu/ml to 3.67×105 cfu/ml and 3.6×108 cfu/ml to 2.33×106 cfu/ml, respectively under 4°C and room temperature (RT), from 0 days after inoculation (DAI) to 90 DAI. Similarly, for solid formulation, the bacterial population declined from 3.33×108 cfu/ml to 3.0×105 cfu/ml and 3.33×108 cfu/ml to 2.0×105 cfu/ml, respectively. The two bioformulations of P. luminescens were evaluated against T. truncatus on cucumber in polyhouse. By the 7th day of treatment, the liquid formulation (99.15 %) resulted in significant reduction in the mite population comparable with that of spiromesifen (99.92%), horticultural mineral oil (99.76 %) and neem oil emulsion (98.02 %), followed by solid bioformulation (94.61 %). The research identified two native isolates of P. luminescens possessing significant acaricidal activity against T. truncatus. The effectiveness of both liquid and solid formulations of P. luminescens brought out in the study suggests the potential of this bacterium for utilization in mite pest management.Item Insecticide tolerance in stingless bee, Tetragonula iridipennis Smith(Hymenoptera: Apidae: Meliponini)(Department of Agricultural Entomology, College of Agriculture ,Vellanikkara, 2023-12-08) Vineetha, V.; Mani ChellappanBees are the most ecologically and economically important group of insects. Many cross-pollinated crops rely on bees for pollination services. Among the bees, stingless bees (Apidae: Meliponini) form the largest group with over 50 genera and more than 505 known species. Stingless bees play a prominent role in the pollination of many food crops. They are recognized as viable pollinators in cucurbits due to their remarkable biological traits that make them suitable for supervised pollination. Stingless bees regularly visit a wide range of flowering crops and often encounter various agrochemicals while foraging in agro-ecosystems. Continuous exposure to insecticides in crop ecosystems can lead to insecticide tolerance in bee populations, much like in pestiferous insects. However, the different mechanisms underlying pesticide detoxification in stingless bees have gained little attention so far. In this context, the present study was undertaken with the objectives of assessing the toxicity of different insecticides to stingless bees, detecting insecticide residues in pollen, determining the activity of metabolic enzymes and investigating the role of gut endosymbionts in insecticide detoxification. Purposive surveys were carried out to document the information on insecticide use in cucurbits from four districts of Kerala viz., Malappuram, Palakkad, Thrissur and Ernakulam. Survey among 120 farmers in four districts recorded the use of 26 different insecticides in cucurbits on an average of 5 to 7 sprays per season at an interval of 12 to 15 days. Based on the survey, the most widely used insecticides viz., chlorantraniliprole, thiamethoxam, malathion, flubendiamide and dimethoate were selected for further toxicological bioassays. Toxicological assays were carried out in two populations of stingless bees collected from a feral colony and an agro-ecosystem using a new protocol standardized as part of the study. The results of bioassay showed that stingless bees from the feral colony had low toxicity to chlorantraniliprole (LC50 of 12.53 ppm) followed by flubendiamide (11.50 ppm), dimethoate (5.28 ppm), malathion (1.20 ppm) and thiamethoxam (0.37 ppm). In comparison, the insecticide flubendiamide was least toxic (LC50 of 20.58 ppm) to stingless bees from the agro-ecosystem followed by chlorantraniliprole (15.59 ppm), dimethoate (9.52 ppm), malathion (4.02 ppm) and thiamethoxam (0.78 ppm). A field experiment was conducted to investigate the presence of insecticide residues in bee pollen by raising bitter gourd plants and fixing stingless bee hives in each treatment plot. Insecticides were sprayed onto the treatment plots and bee pollen samples were collected for residue analysis. The LCMS/ MS and GC analysis of pollen samples detected residues of fipronil and chlorantraniliprole at concentrations of 0.596 mg/kg and 0.108 mg/kg, respectively. The biochemical analyses of stingless bees collected from different treatment plots on 0, 1, 3, 5 and 7 days after spraying insecticides revealed that the total protein content in stingless bees ranged between 2.307 mg/ml for bees in the control plot and 2.565 mg/ml in bees from the dimethoate-treated plot. Highest protein concentration was observed on the third day after treatment application. The highest activity of the detoxifying enzyme, carboxyl esterase was observed in bees collected from the dimethoate-treated plot (83.024 μMol min-1 mg protein-1) and the least in bees from the control plot (26.599 μMol min-1 mg protein-1). The production of carboxyl esterase was found to be highest on the 5th day after spraying. The treatment-wise comparison of the detoxifying enzyme, cytochrome P450 in stingless bees revealed that the enzyme activity was significantly higher in bees collected from chlorantraniliprole treated plot (383.960 pMol min-1 mg protein-1) compared to the control (4.926 pMol min-1 mg protein-1). The cytochrome level was significantly higher on 3rd day after spraying of insecticides. Higher glutathione Stransferase (GST) was induced in bees when sprayed with dimethoate (382.206 μMol min-1 mg protein-1), whereas the level of GST in bees in the control plot was very low (3.164 μMol min-1 mg protein-1). The highest GST activity was observed one day after treatment application which differed noticeably among other days. Investigation and comparison of the gut microbiota of stingless bees collected from the forest and agro-ecosystem were done through metagenomic DNA isolation and next-generation sequencing (NGS) technology. The metagenomic sequencing results showed the presence of 103763 microbes belonging to 123 species and 41080 microbes from 272 species in the gut of stingless bees from the forest and agricultural ecosystems respectively. The predominant microbial species recorded in the bee gut of forest ecosystem was Klebsiella sp. (70.26 %) and in agro-ecosystem was Pantoea agglomerans (84.68 %), which are reported to be involved in insecticide degradation followed by chlorantraniliprole (15.59 ppm), dimethoate (9.52 ppm), malathion (4.02 ppm) and thiamethoxam (0.78 ppm). A field experiment was conducted to investigate the presence of insecticide residues in bee pollen by raising bitter gourd plants and fixing stingless bee hives in each treatment plot. Insecticides were sprayed onto the treatment plots and bee pollen samples were collected for residue analysis. The LCMS/ MS and GC analysis of pollen samples detected residues of fipronil and chlorantraniliprole at concentrations of 0.596 mg/kg and 0.108 mg/kg, respectively. The biochemical analyses of stingless bees collected from different treatment plots on 0, 1, 3, 5 and 7 days after spraying insecticides revealed that the total protein content in stingless bees ranged between 2.307 mg/ml for bees in the control plot and 2.565 mg/ml in bees from the dimethoate-treated plot. Highest protein concentration was observed on the third day after treatment application. The highest activity of the detoxifying enzyme, carboxyl esterase was observed in bees collected from the dimethoate-treated plot (83.024 μMol min-1 mg protein-1) and the least in bees from the control plot (26.599 μMol min-1 mg protein-1). The production of carboxyl esterase was found to be highest on the 5th day after spraying. The treatment-wise comparison of the detoxifying enzyme, cytochrome P450 in stingless bees revealed that the enzyme activity was significantly higher in bees collected from chlorantraniliprole treated plot (383.960 pMol min-1 mg protein-1) compared to the control (4.926 pMol min-1 mg protein-1). The cytochrome level was significantly higher on 3rd day after spraying of insecticides. Higher glutathione Stransferase (GST) was induced in bees when sprayed with dimethoate (382.206 μMol min-1 mg protein-1), whereas the level of GST in bees in the control plot was very low (3.164 μMol min-1 mg protein-1). The highest GST activity was observed one day after treatment application which differed noticeably among other days. Investigation and comparison of the gut microbiota of stingless bees collected from the forest and agro-ecosystem were done through metagenomic DNA isolation and next-generation sequencing (NGS) technology. The metagenomic sequencing results showed the presence of 103763 microbes belonging to 123 species and 41080 microbes from 272 species in the gut of stingless bees from the forest and agricultural ecosystems respectively. The predominant microbial species recorded in the bee gut of forest ecosystem was Klebsiella sp. (70.26 %) and in agro-ecosystem was Pantoea agglomerans (84.68 %), which are reported to be involved in insecticide degradationItem Biosystematics studies on stingless bees (Apidae : Meliponini) of India(Department of Agricultural Entomology, College of Agriculture , Vellayani, 2024-07-12) Arya Krishna; Shanas, SThe study entitled ‘Biosystematic studies on Stingless bees (Apidae: Meliponini) of India’ was carried out at the College of Agriculture, Vellayani, during 2019-2023 to study the stingless bee diversity in India through morphological and molecular characterisation and document the reproductive biology of Tetragonula travancorica. Extensive sampling was done, and a total of 70 samples were collected from 22 States and 2 Union Territories of India. Samples were collected from domesticated as well as feral colonies by post and field visits. Thirty-four samples were collected through the post and 36 samples were collected through field visits among which, 50 samples were collected from feral colonies and 20 samples from domesticated colonies. 47 per cent of the feral colonies were collected from building basements, 33% from tree hollows, 11 % from rock crevices and 6% from pipes. The samples were processed and preserved for the study of external morphological characters like general shape, size, colour, pilosity, characters of wings, head and thorax. Measurements were made with a calibrated ocular micrometer and pertinent ratios were worked out. Three fifty specimens out of the 70 populations of stingless bee workers were examined. Male genitalia was dissected as per standard protocol and male metasomal sternum 5 and sternum 6 were examined and studied wherever males could be collected. 31 morphological measurements were used in the Principal Component Analysis (PCA) revealing three clusters corresponding to three genera of stingless bee populations viz., Tetragonula, Lisotrigona and Lepidotrigona, identified based on the key to the genera. Species identity was confirmed based on the characters of male genitalia, metasomal sternum and comparative morphometric ratios, obtained from the available literature. Diagnosis of all identified species and detailed descriptions of the new species are provided. The samples collected from all over India could be identified as T. srikantanathi, T. callophyllae, T. travancorica, T. ruficornis, T. shishirae, T. shubhami, Lepidotrigona arcifera and Lisotrigona chandrai. Two putative new species have been identified from Tamil Nadu and Andaman and Nicobar Islands. T. callophyllae was identified from Goa which is the first report of this endangered stingless bee from outside Kerala. Males of T. travancorica were described for the first time. The reproductive biology of T. travancorica was studied using an acrylictopped wooden hive box of 12x4x4 inch size. The length, width, shape and colour of egg and brood cells were recorded. The average length and width of the eggs were found to be 0.88mm and 0.32 mm respectively. The eggs were oval shaped and their colour ranged from transparent to light white. The average length and width of the brood cells were found to be 4mm and 3mm. The brood cells had an oval shape and their colour ranged from dark brown to whitish brown. T. travancorica was found to have an egg period of 4-5 days, a larval period of 18-19 days and a pupal period of 20- 24 days. The total development period was found to be 42-48 days. The oviposition time was found to be 4-7 seconds and the brood cell closing time was found to be 4-6 minutes. Molecular barcodes of the two putative new species and T. callophyllae were generated. Twenty-five gene sequences of stingless bees were obtained based on which, a phylogenetic tree was derived. It showed that the Indian population of stingless bees formed 2 main clades. The first clade included populations belonging to the Genus Tetragonula. The second main clade forms two subclades. The first subclade included T. callophyllae and Lisotrigona chandrai indicating a common ancestor. The second subclade again forms two clusters in which the first includes the populations of stingless bees from Andaman and Nicobar Islands including the putative new species from Andaman and Nicobar Islands. The second cluster includes the population of stingless bees representing the genus Lepidotrigona indicating the presence of a common ancestor. The stingless bees of India comprise 27 species placed in 3 genera. A checklist of Stingless bees in India is provided. The study treated 8 species under 3 genera, 2 putative new species, a new distributional record of T. callophyllae from Goa and a description of males of T. travancorica for the first time. The reproductive biology of T. travancorica was documented. Morphological and molecular characterisation was attempted and a phylogenetic tree was derived and PCA was carried out to clarify the present status of stingless bee taxonomyItem Characterization of microbiota associated with stingless bee Tetragonula travancorica Shanas and Faseeh(Hymenoptera:Apidae:Meliponinae) and its pollen(Department of Agricultural Entomology, College of Agriculture,Vellanikkara, 2024-09-12) Bindu,G R.; Mani ChellappanStingless bees are small to medium-sized eusocial insects belonging to the tribe Meliponini (Hymenoptera: Apidae). They are one of the potential pollinators in the tropics and adapted to varied climatic conditions than other pollinators such as honey bees and bumble bees. There are over 600 species of stingless bees worldwide, with India recording three genera viz., Tetragonula Moure, Lisotrigona Moure, and Lepidotrigona Schwarz. Tetragonula is the most complex and widely distributed genus with more than 30 and 17 species worldwide and in India, respectively. A new species was discovered in Kerala, Tetragonula travancorica Shanas and Faseeh, and found to be the widespread species of stingless bee in peninsular India. Stingless bees and their products host symbiotic microbes, which play a prominent role in metabolism, hormonal signaling, behaviour, gut physio-chemical conditions, growth and development, protection against pathogens, and the immune response of bees. However, the microbial communities associated with stingless bees and their pollen remain poorly understood. Hence, the present research programme was proposed to study the pollen sources, and microbiota associated with stingless bees, hive-stored pollen, and flower pollen. A survey was carried out to collect stingless bees (T. travancorica) and their hive-stored pollen from all districts of Kerala for palynological and microbial diversity analysis. Palynological studies conducted through light microscopy and scanning electron microscopy revealed the presence of 102 pollen types from 38 plant families. Eight predominant pollen types were found across Kerala, viz., Mimosa pudica, Mangifera indica, Coffea arabica, Bauhinia purpurea, Alternanthera sessilis, Pennisetum polystachion, Artocarpus heterophyllus, and Cocos nucifera. The hivestored pollen analysis of stingless bee T. travancorica recorded the highest number of pollen types from the family Fabaceae (14) followed by Asteraceae (9) and Euphorbiaceae (6). The maximum pollen types belonged to the trees followed by weeds, ornamentals, and horticultural crops. Absolute pollen count was the highest in Kannur district (11,01,000 pollen grains/mL) and lowest in Idukki district (1,28,500 pollen grains/mL). The microbial diversity analysis of hive-stored pollen revealed the presence of bacteria and fungi, whereas, yeast and actinomycetes were not detected across Kerala. A total of 24 bacterial and 20 fungal isolates were identified in the hive-stored pollen and their morphological, and cultural characteristics were recorded. Among them, distinct bacterial isolates were subjected to biochemical characterization, which revealed that all bacterial isolates showed positive for the catalase test, while six isolates were positive for the oxidase test. All the isolates except one isolate were negative for the methyl red test and five isolates were positive for the Voges Proskauer test. Seven isolates showed positive reactions for sucrose and fructose fermentation, which helps in identifying bacterial isolates. Molecular characterization of bacterial isolates revealed that Bacillus spp., B. cereus, B. acanthi, B. velezensis, B. subtilis, B. megaterium, Priesta aryabhattai, Pseudomonas spp., P. aeruginosa, Klebsiella pneumonia, and Acinetobacter spp., were recorded in the hive-stored pollen of stingless bee. Fungal isolates viz., Aspergillus flavus, A. aculeatus, Penicillium spp., and P. chrysogenum were identified in the hive-stored pollen of stingless bees. The microbial load to pollen grain ratio was highest in Idukki (1:30.5) and lowest in Kollam (1:25750). The highest total bacterial and fungal population was observed in Idukki (4160 cfu g-1 of pollen), Pathanamthitta, and Thrissur (90 cfu g-1 of pollen). The lowest bacterial and fungal population was recorded in Kollam and Alappuzha (10 cfu g-1 of pollen), respectively. The predominant pollen and secondary pollen obtained from the various districts of Kerala were selected to study flower microbial diversity. The microbial analysis of flower pollen revealed the presence of bacteria and fungi, whereas yeast and actinomycetes were not detected in the flower pollen. A total of 19 bacterial and 21 fungal isolates were recorded in the flower pollen and identified with morphological and cultural characteristics. Biochemical tests revealed that all the isolates were catalase-positive and four being oxidase-positive. Six bacterial isolates tested positive for the methyl red test, while five were positive for the Voges- Proskauer test. Eight isolates showed sucrose utilization, indicated by a color change from red to yellow without air bubbles in the durum tube. Molecular characterization of the bacterial isolates revealed that Priesta aryabhattai, Bacillus megaterium, B. safensis, B. pumilis, Pseudomonas aeruginosa, Pantoea spp., P. dispersa, and Serratia marcescens were present in the flower pollen. Similarly, molecular characterization of fungal isolates revealed that Penicillium spp., P. chrysogenum, P. citrinum, Curvularia warraberensis, C. clavata, Aspergillus spp., A. niger, A. aculeatus, A. flavus, Pestalotiopsis spp., Fusarium incarnatum, and Mucor irregularis were present in the flower pollen. The microbial load-to-pollen grain ratio was highest for Pennisetum polystachion (1: 0.84), and the lowest ratio for Mimosa pudica flower pollen(1:8.8), Alternanthera sessilis had the highest total bacterial population (63,000 cfu g-1 of pollen), while Mimosa pudica had the lowest (5000 cfu g-1 of pollen). Similarly, Areca catechu had the highest total fungal population (90 cfu g-1 of pollen), while Pennisetum polystachion had the lowest (30 cfu g-1 of pollen). The gut microbiota of stingless bees, T. travancorica, and their hive-stored pollen were investigated and compared through metagenomic analysis under a nextgeneration sequencing platform. The sequence analysis revealed that Lactobacillus (41.57 %) was the dominant symbiont in the gut of T. travancorica, while Bacillus (28.11 %) was the predominant genus in the pollen of T. travancorica. Similarly, the fungal symbionts associated with the gut of T. travancorica revealed that Rhodosporidiobolus (15.46 %) was the dominant fungal symbiont in the gut, while Saccharomycopsis (27.67 %) was dominant in the pollen. Characterization of the microbiota associated with the stingless bees and their pollen unveils the complex relationship between bees and their microbial symbionts. Bee and its products are potential sources of beneficial microorganisms that have wide applications in various fields. The specific microbiota associated with the bees helps to enhance beekeeping practices, reducing colony losses and aiding in the conservation of these crucial pollinators. Harnessing these bees and their symbionts has an immense scope in agriculture, food, and probiotic industriesItem Population dynamics and management of rice yellow stem borer Scirpophaga incertulas (Walker) and leaf folder Cnaphalocrocis medinalis (Guenee) in South Kerala(Department of Agricultural Entomology, College of Agriculture , Vellayani, 2022-12-19) Sharanappa.; Suja, GThe present study entitled “Population dynamics and management of rice yellow stem borer, Scirpophaga incertulas (Walker) and leaf folder, Cnaphalocrocis medinalis (Guenee) in South Kerala” was carried out at Department of Agricultural Entomology, College of Agriculture-Vellayani, from 2018 to 2021. A survey was conducted in major rice growing areas viz. Thiruvananthapuram, Onattukara and Kuttanad to study the population dynamics of rice stem borers and leaf folders in South Kerala in relation to biotic and abiotic factors. Field tolerance of different released varieties against rice stem borers and leaf folders and management studies were conducted at Onattukara Regional Agricultural Research Station, Kayamkulam. In addition to the common species of stem borer and leaf folder in rice, the white stem borer S. innotata was recorded from Onattukara and Kuttanad and the leaf folder species M. patnalis from Thiruvananthapuram and Onattukara. The lowest stem borer and leaf folder damage in rice was recorded in Kuttanad at 75 and 90 DAS respectively in southern Kerala. The major natural enemies recorded were the predators viz. dragonflies, damselflies, coccinellids and spiders and parasitoids viz.Cotesia sp, Isotima sp. and Tetrastichus sp. The rice varieties Samyuktha and Onam recorded significantly low stem borer infestation and Makom significantly low infestation by leaf folders. The highest phenol content was estimated in the rice variety Onam both at vegetative and flowering stages and highest silica content in Supriya and Sreyas. The bio-pesticides viz. 1% azadirachtin 0.003%, dasagavya 3%, cashewnut shell liquid 0.1% and T. japonicum and T. chilonis each 1 lakh ha-1 are effective in controlling stem borer and leaf folder infestation and safe to natural enemies. Significantly high grain yield and highest marginal benefit: cost ratio was recorded in the treatments chlorantraniliprole 18.5 SC 0.005% and 1% azadirachtin 0.003%. This study leads to the future investigations on identification of new species of stem borers, leaf folders and associated natural enemies. Breeding programmes can be taken up with tolerant varieties and management of these pests with new bio-pesticides can be done.Item 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 BEnvironmental 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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