Endophytic entomopathogenic fungi Akanthomyces lecanii, were found to establish a covert symbiotic association within banana Musa spp. and conferring enhanced resistance against herbivorous pests. This endophytic relationship is a promising, and sustainable approach for integrated pest management in banana cultivation. The banana aphid, Pentalonia nigronervosa, plays a critical role in insect-microbe interactions and serves as a target for aphid control to reduce virus transmission. In this study, Gas Chromatography-Mass Spectrometry (GC-MS) was employed to identify volatile organic compounds (VOCs) produced by P. nigronervosa and to characterize the metabolic repertoire of A. lecanii. Endophytic fungi were isolated from pseudostem tissues, which yielded multiple fungal isolates, were taxonomically characterized through sequencing the internal transcribed spacer (ITS) region. Subsequently, bioassays were conducted to assess their effect on aphid survival and behavior. GC-MS metabolomic profiling revealed that both Akanthomyces lecanii and Pentalonia nigronervosa produces a range of volatile organic compounds including Triphenyl phosphate, p-Cymen-7-ol (4-Isopropylbenzyl alcohol), Valeric acid, 4-Ethylbenzaldehyde (Caproic Acid), Pyridoxal phosphate, Palmitic Acid, 2-Methyloctan-3-ol and Ethanone (Acetaldehyde) several of the bioactive compounds exhibit aphicidal properties with potential insect-repellent effects. Notably, 2-Methyloctan 3-ol, ethanone, Palmitic acid, and Pyridoxal phosphate emerged as the most potent aphicidal candidates and were formulated into eco-friendly insecticidal blends. Overall, our results highlight the chemical ecology of aphid-microbes interaction and provide a metabolomic-guided foundation for novel phytochemical strategies to protect banana crops from aphid-borne diseases.

Lichens, a diverse group of Ascomycetes within the major fungal phylum Lecanorales, include various forms such as Crustose, Foliose, Squamulose, and Fruticose, with the latter being the most common photobionts. This study aims to provide novel in-sights into the extraction and acquisition of bioactive compounds from lichens, emphasizing their potential for future advancements in analytical techniques. This research highlights the pharmaceutical potential of secondary metabolites from lichens, with a focus on GC-MS analysis, which identified 80 compounds belonging to various functional groups. Many lichens have demonstrated bioactive properties, including novel drug candidates such as Falcarinol, Oleic Acid methyl ester, Benzoic acid, 2,4-dihydroxy-3,6-dimethyl, Glycerol 1-palmitate, Guanosine, 2(3 H)-Furanone, 3-butyldihydro, Eudesma-5,11(13)-dien-8,12-olide, Phthalic acid butyl undecyl ester, Phthalic acid ethyl pentadecyl ester, Tridecanoic acid 12-methyl methyl ester, Undecanoic acid 10-methyl methyl ester, Aromandendrene, Dodecahydroacenaphthylene, 3-Bromo-7-methyl-1-adamantanecarboxylic acid, Estra-1,3,5(10)-trien-17 beta-ol, 3-Methoxy-5-propylphenol, and Phenol. Consequently, P. aurata and P. reticulatum exhibited the highest antimicrobial activity, effectively inhibiting bacterial strains such as E. coli, K. pneumoniae, and S. aureus, particularly in terms of zones of inhibition. Additionally, the lichen extracts showed notable anti-tumor effects against HT-29 colon cancer cells, demonstrating a clear dose-dependent response for (10 mg/mL). Novel scientific methodologies involving biological entities have significantly contributed to a deeper understanding of lichenology. The research hypotheses are: (a) the collection of higher fungi, selective isolation of lichens, in vitro culture, and conservation; (b) extraction of lichen secondary metabolites and GC-MS analysis using innovative bioactive techniques.

The rugose spiralling whitefly, Aleurodicus rugioperculatus is an invasive phloem-feeding pest and acts as vector for virus transmission in banana, coconut, and other economically important crops in southern India. The main aim of this research work was to evolve a novel and eco-friendly strategy to combat this serious pest. The entomopathogenic bacterium Bacillus thuringiensis produces parasporal crystal proteins, commonly known as δ-endotoxins and widely exploited as eco-friendly insecticides. This study tested two native B. thuringiensis strains namely NBAIR-TrBt18 and NBAIR-Bt25 that were confirmed to carry the Cry16A and Cry1Ab genes. The insecticidal efficacy for volatile organic compounds emitted through insect-microbes interaction as well as under natural conditions were analysed. Bioassay of purified parasporal crystals against pre-instar nymphs showed significant mortality at lower concentrations of 2.0 µg/mL (NBAIR-TrBt18) and 2.7 µg/mL (NBAIR-Bt25) at 72h. GC-MS analysis of the headspace volatiles revealed several bioactive compounds including Z-10-Tetradecen-1-ol acetate (10Z)-10-Tetradecenyl acetate), Trans-Z-α-Bisabolene epoxide, Phenylethyl alcohol, 7-Hexadecenal, (Z)-, 2-Ethyl-1-hexanol, 2-Butanone, 2-Heptanone, Benzaldehyde, Isovaleric acid, Nonanoic acid, Oleic Acid and Decanal. Twelve detected volatiles were tested for insecticidal activity against A. rugioperculatus. These compounds showed a positive correlation with observed mortality rates. Notably, Z-10-Tetradecen-1-ol acetate (10Z)-10-Tetradecenyl acetate), trans-2-Butanone, and trans-Z-α-Bisabolene were particularly exhibiting the highest mortality at 3rd day post-inoculation on 4th pre-instar nymphs. These findings confirm that (B) thuringiensis strains not only produced potent crystal toxins but also release volatile organic compounds that possessed both insecticidal and insect repellents properties at low dosages. Overall, this research provides a foundation for the development of novel eco-friendly biocontrol formulations by integrating selective metabolites with native B. thuringiensis strains to manage A. rugioperculatus for a sustainable and safe pest management option.

Secondary metabolic compounds were investigated on the various insecticides from plants based on extraction and profile identified on various toxic substances. Generally, interactions between insects and plants lead to the release of various biochemical components pivotal in secondary metabolic processes and in insect defense against stimuli or insecticides. This study highlighted the efficacy of botanical pesticides containing bioactive chemicals as insecticides, operating through mechanisms such as antifeedants, repellents, protectants, and growth-disrupting hormones. Thus, secondary metabolic activity was confirmed to exhibit insecticidal properties, including the emission of signalling cues such as 2,4-Decadienal, Pterin-6-carboxylic acid, Oleic Acid, 9-Octadecenoic acid (E), and Stearic acid. Meanwhile, Principal component analysis was used to assessed the distribution of metabolites resulting from plant-insect interactions. Additionally, putative toxic substances were confirmed in repellent assays, affirming that botanical blends enhanced certain plant defenses against banana pests. Further understanding of these components and their varying efficacy levels under different conditions may be crucial in developing bio-rational control against B. subcostata under in vitro conditions. The current study aims to examine certain plant extracts as natural enemies and alternatives against the banana fruit scarring beetle B. subcostata under in vitro conditions.

IRF9 is a crucial component in the JAK-STAT pathway. IRF9 interacts with STAT1 and STAT2 to form IFN-I-stimulated gene factor 3 (ISGF3) in response to type I IFN stimulation, which promotes ISG transcription. However, the mechanism by which IFN signaling regulates Malabar grouper (Epinephelus malabaricus) IRF9 is still elusive. Here, we explored the nd tissue-specific mRNA distribution of the MgIRF9 gene, as well as its antiviral function in E. malabaricus. MgIRF9 encodes a protein of 438 amino acids with an open reading frame of 1317 base pairs. MgIRF9 mRNA was detected in all tissues of a healthy M. grouper, with the highest concentrations in the muscle, gills, and brain. It was significantly up-regulated by nervous necrosis virus infection and poly (I:C) stimulation. The gel mobility shift test demonstrated a high-affinity association between MgIRF9 and the promoter of zfIFN in vitro. In GK cells, grouper recombinant IFN-treated samples showed a significant response in ISGs and exhibited antiviral function. Subsequently, overexpression of MgIRF9 resulted in a considerable increase in IFN and ISGs mRNA expression (ADAR1, ADAR1-Like, and ADAR2). Co-immunoprecipitation studies demonstrated that MgIRF9 and STAT2 can interact in vivo. According to the findings, M. grouper IRF9 may play a role in how IFN signaling induces ISG gene expression in grouper species.

Pr1A a subtilisin-like protease was generated by Metarhizium is known to involved in the early stages of fungal invasion of arthropod hosts. Current intended to establish a link between cotton bollworm mortality H. armigera expression on levels of the conidial pr1A gene in Metarhizium. Intriguingly, subtilisin-like proteases were emitted by defensive metabolism on volatile organic compounds in H. armigera products. Fifth day after treatment, conidial of M. anisopliae, M. robertsii, M. majus (6.59, 4.65, and 4.65 × 105 conidia/ml) respectively, resulted in greater than 95

Background Among all the coconut pests, rhinoceros beetle causes acute and serious damage to coconut palm. Management of this pest is very difficult due to its nocturnal activity, and also, it damages the emerging leaf inside the bud. Management of rhinoceros beetle using entomopathogens will be of great importance as it is economical and ecofriendly. Studies were carried out to decipher the biocontrol potential of indigenous entomopathogenic bacterium ( Bacillus thuringiensis ) and entomopathogenic fungi ( Metarhizium robertsii and M. majus ) against Oryctes rhinoceros in the field, individually as well as in combination, by soil drench as well as by topical spray method. Results The study showed that B. thuringiensis strain NBAIR-BTAN4 showed 24.8% mortality at 5th week in soil drench method and 24% mortality in topical spray method. M. robertsii showed 24% mortality at 5th week in soil drench method as well as topical spray method. Similarly, M. majus showed 24% mortality in soil drench method and 23.2% in topical spray method at 5th week. In combination, NBAIR-BTAN4 + M. robertsii showed 40.8 and 44% in soil drench and topical spray method at 5th week, respectively. Combination of NBAIR-BTAN4 + M. majus showed 44.8 and 40.8% in soil drench and topical spray method at 5th week, respectively. Combination of NBAIR-BTAN4 + M. robertsii + M . majus showed 52.8 and 57.6% mortality at 5th week in soil drench and topical spray method, respectively. Conclusion The present study showed application B. thuringiensis in combination with M. robertsii and M . majus is effective in management rhinoceros beetle in coconut orchard. Study also indicated that soil drench method is more promising strategy than topical spray method in managing larval population of the beetles and also confirmed that B. thuringiensis , M. robertsii and M. majus are compatible and they seemed to have a synergistic effect in controlling the pest in coconut orchard.

Background The banana stem weevil, Odoiporus longicollis (Olivier), is a serious threat to banana cultivation world over. Since banana is a food crop, the use of naturally infecting biological control agents could be an effective alternative to manage the insect pest instead of harmful chemicals. Also, the efficacy of entomopathogenic fungi against O. longicollis was used in bioassay. Results Among the Beauveria bassiana isolates tested the median lethal concentration (LC 50 ) 10.468 × 10 5 conidia ml −1 when treated with B. bassiana (NRCBEFPMP1), two other isolates of B. bassiana , namely NRCBEPF22 and NRCBEPF2, were also effective against O. longicollis and recorded LC 50 of 12.617 × 10 5 and 12.891 × 10 5 conidia ml −1 , respectively. The results of bioassay with different Metarhizium spp. showed variations in efficacy, where the most virulent isolate was M. quizhouense (NRCBEPF11) with LC 50 8.050 × 10 5 conidia ml −1 . Scanning electron microscopic analysis showed that B. bassiana and M. quizhouense caused infection by cuticle penetration and completed the infection process in 15 days. The composition of volatile organic compounds released by B. bassiana and M. anisopliae during pathogenesis showed that a significantly high number of known insect volatiles were present in infected insects. Consequently, these volatiles were emission in Insect attractant, Odorant receptor agonist, Plant hormone Plant, and Microbial Metabolites, through the biological activity, such as Methyl salicylate, Benzaldehyde, alpha-Terpineol, Limonene, Benzene, 1,2-dimethoxy, Phthalic acid, 1-Octadecene, Phenylacetaldehyde, 3-Octanone, Octanal, Methylheptenone and 2-Ethyl-1-hexyl alcohol. Conclusion Overall, the results show that EPF could significantly reduce damage by O. longicollis and produce a wide profile of secondary metabolites. Further, analysis was used for principal components to determine whether separated classes of fungi can be distinguished from one another based on their metabolite profiles.

A field survey was done in teak (Tectona grandis F.) forests in South India to explore the entomopathogenic effect of Metarhizium anisopliae (Ascomycota: Sordariomycetes) against teak defoliator, Hyblaea puera (Lepidoptera: Hyblaeidae). About 300 soils and infected insect samples were collected during the survey and thirty-six fungal isolates were isolated from soil and insect samples and characterized. The fungi were cultured on PDAY with dodine and antibiotics. Generally, the EPF culture was incubated at 27 °C in darkness for 15 days. Virulence of the Entomopathogenic Fungi (EPF) ability to germinate under cold and heat temperatures was assessed in a culture impregnated with conidia. In the experiment, it was found that for the first time Metarhizium quizhouense, Metarhizium robertsii, and Metarhizium majus species caused significantly higher mortality to hosts. These isolates of M. anisopliae, M. robertsii, M. majus, and M. quizhouense were all considered to be effective virulent and environmentally adaptive. The Metarhizium isolates were recommended as effective bio-control agents through the field investigation of teak defoliator Hyblaea puera from South India forest. This study paves the way to utilize the indigenous isolates of EPF for the control of teak defoliator and to combat the pests thatare resistant to insecticide.

Background Banana fruit scarring beetle (BFSB), Basilepta subcostata (Jac.) (Chrysomelidae:Coleoptera), is an important insect pest feeds on leaf and fingers, which affects the cosmetic value of the fruit. The pest is distributed in Assam, Bihar, West Bengal, Chhattisgarh, and North-eastern Hill regions of India. Results The pest is currently managed by foliar spray with insecticides. In order to identify eco-friendly control of the pest, attempts were made to isolate microbial agent and evaluate their potential to control the pest. A total of 27 entomopathogenic fugal isolates were obtained from Odoiporus longicollis (Oliver), Cosmopolites sordidus (Germar), Basilepta subcostata (Jac), and other insect Galleria mellonella (Fabr) . Based on colony morphology, the collected fungal isolates were identified as Metarhizium spp. (17) and Beauveria spp. (10). Through ITS sequencing, the fungal isolates were further characterized at species level as B. bassiana (8), B. brongniartii (2), M. anisopliae (8), M. robertsii (6), M. guizhouense (2), and M. pinghaense (1). Their sequences were submitted in GenBank and obtained accession numbers. Among 27 isolates tested against B. subcostata under laboratory conditions, 3 isolates ( M. anisopliae NRCBEPF-36, M. pinghaense NRCBEPF-7 and B. brongniartii NRCBEPF-27) recorded 100% beetle mortality, followed by 11 isolates with 95–99% and 13 isolates with 88–93% within 8 days of treatment. Conclusion This study highlights the two native North East India isolates B. brongniartii NRCBEPF-27 (MT151781) and M. anisopliae NRCBEPF-36 (MT140308) showed the significance to use as potential biocontrol agents against banana fruit scarring beetle B. subcostata. Further experiments under field conditions are required to evaluate their biological control efficacy against the pest .

The search for new Bacillus thuringiensis ( Bt ) strains is a continuous process and researchers are now focusing on finding toxin proteins that are toxic to pests of insect orders that are not reported. In the present study soil and insect cadaver samples were collected from North East India comprising the states of Assam, Tripura and Mehhalaya and native Bt were isolated using standard protocols. At total of 30 Bt isolates were purified and characterized. Various types of crystal morphology were encountered that included bipyramidal, cuboidal, square, rhomboid, spherical and irregular. PCR analysis showed that diverse cry genes were expressed. The cry genes identified were Lepidoptera, Coleoptera and Diptera specific. Detected genes included cry1Ac , cry2A , cry4A , cry10A , cry16A , cry17A , cry19A , cry30Aa , cry44Aa , cry11A , cry4B , cry12A , cry8A and cry7A . Many of them were positive for Vip3A protein. The coleopteran specific Bt were evaluated against Sitophilus oryzae and Callosobruchus chinensis and NBAIR-AgBt6 was found to be toxic. The isolates are being further evaluated for use as biopesticides.

Studies were conducted to systematically isolate Metarhizium isolates from the insect cadavers and soils of South India. Morphological and PCR amplified sequences of 5.8S ITS regions and RNA polymerase II largest subunit (RPB1) gene regions were used to identify the isolates at species level. Eight Metarhizium isolates were isolated and initially identified by morphological and microscopic studies. Further identification was confirmed through 5.8SrRNA ITS and RPB1 analysis. They were identified as three isolates of M. robertsii J.F. Bisch., Rehner & Humber sp. nov. (ArMz3R, ArMz3S and ArMz6W), one isolate of M. majus (J.R. Johnst.) J.F. Bisch., Rehner & Humber (VjMz1W) and four isolates of M. anisopliae (WnMz1S, NlMz2S, BgMz2S and DhMz4R). Topical conidial suspensions (TCS) and powder based formulations (PBF) of the eight indigenous isolates of Metarhizium spp. that were isolated from insect cadavers and soils of South India were tested against coleopteran pests Holotricha serrata L. and Oryctes rhinoceros L. that cause serious damage to sugarcane and palm trees respectively. Against H. serrata TCS of M. robertsii (ArMz6W) was the most effective with an LC50 of 6.893í—105 cfu/ml and caused 100% mortality against the 3rd instar larvae in 5 days; PBF elicited an LC50 of 7.502í—105 cfu/ml with 96% mortality in 10 days. Against O. rhinoceros TCS (LC50 of 9.75í—105 cfu/ml) of M. majus (VjMz1W) caused 90% mortality in 7 days and the PBF (LC50 of 9.57í—105 cfu/ml) caused 86% mortality in 14 days. The results establish that M. robertsii is highly effective against H. serrata and against O. rhinoceros, M. majus was the most effective. The TCS formulations of these two strains can be readily deployed for field applications.

The present study was undertaken to test vip3A protein from Bacillus thuringiensis for use against lepidopteran pests especially Spodoptera litura and whether cloning and expression of the vip3A gene will help in formulating an improved bioinsecticide. Eight isolates that showed amplification of the vip3A gene (675bp) were selected from 150 B. thuringiensis isolates. The isolate BT-EG1 was selected for further studies as the vip3A protein extracts from this isolate was found to cause mortality of Spodoptera litura (LC 50 of 9.09-9.92 µg/ml). The 2.367bp complete CDS was amplified using VCL1 and VCL2 specific primers and cloned into P UC19 at NdeI and XhoI restriction sites. The positive clones were sequence confirmed. The vip3A was the successfully cloned into pET21a (NdeI/XhoI) expression vector and confirmed by restriction digestion and SDS-PAGE. The crude protein extracts obtained after 16h of IPTG induction showed a LC 50 of 5.83 µg/ml against S. litura at 72h . The extracts were however highly toxic to Plutella xylostella (LC 50 of 0.43 µg/ml after 16h of IPTG induction at 48h). The extracts however did not cause mortality of Helicoverpa armigera suggested the requirement of a combination of vip proteins for broad spectrum activity

ABSTRACT: HCry gene and plasmid profiling of indigenous Bacillus thuringiensis isolates from North East India and Andaman were carried out. A total of 29 isolates were screened and HD-1 was used as reference. Plasmid profiling showed distinct bands of different sizes with unique patterns for each strain. Twenty one isolates had plasmids above 33500 bp and only 14 had plasmids of the same size. One isolate NBAII-TRBT17 showed presence of four plasmids having sizes of 2500bp, 7000bp, 7500bp and 33500bp.Six of them had three plasmids of different sizes. The isolates NBAII-BT5, NBAII-TRBT9, NBAII-TRBT18, NBAII-ASBT15, NBAII-ASBT11 andNBAII-AGBT5 showed similar band migration with three plasmids between 7000 to 9000 bp and 33500 megaplasmid each. Strain NBAII-BTN3 showed two different plasmids but plasmid size ranged as 9000 bp and 33, 500 bp which had similar pattern with NBAIIASBT1, but NBAII-ASBT1 also harbored plasmid above 33500 bp. The isolates NBAII-TRBT10, NBAII-TRBT16, NBAII-TRBT8, NBAII-BTAN$,NBAII-BTAN5, NBAII-ASBT20, NBAII-ASBT2, NBAII-ASBTN1, NBAII-BTEG1, NBAII-AGBT13,NBAII-AGBT6, NBAII-AGBT1 and NBAII-ASBT21 showed presence of a single plasmid above 33500bp. The isolates NBAII-ASBT24, NBAII-BT3 and NBAII-AGBT25 showed similar migration of plasmids ranging between 15000-33500 bp. The plasmids were probed for Cry1, Cry2 and Cry3 genes with universal primers and isolates showed differential expression of the genes. Six of the isolates namely NBAII-ASBT20, NBAII-ASBT24, NBAII-TRBT10, NBAII-TRBT17, NBAII-BTAN4 and NBAII-BTAN5 showed presence of Cry3 gene also apart from Cry1 which could indicate that it could be active against coleopterans also. Plasmid DNA profiles and cry protein characterization provided information on diversity among the isolates and the cry gene diversity.