Development of transgenic cotton (Narasimha) using triple gene Cry2Ab-Cry1F-Cry1Ac construct conferring resistance to lepidopteran pest

Silkworm silk protein fibroin is widely exploited to develop novel silk-based biomaterials due to its stable b-sheetstructure, providing high crystallinity and tensile strength. The polymorphic behaviour of silk fibroin provides awindow to modulate its structural transitions during self-assembly for different functional outcomes. Most studiesare therefore mainly focused on formation of well-developed b-sheet structure and self-assembly of silk fibroinwhich are regulated by many parameters. Glyoxal, a highly reactive a-oxoaldehyde, reacts with different proteinsto form advanced glycation end products (AGEs) following Maillard-like reaction. Considering the significanceof protein modification by glyoxal-derived AGEs, in the present study the effect of glyoxal (250, 500 and1000 lM) on the structure of silk fibroin has been investigated. CD and fluorescence studies reveal that higherconcentrations of the a-oxoaldehyde induce considerable alterations of secondary and tertiary structure of theprotein leading to aggregation following incubation with for 3 weeks. The aggregates exhibit fibrillar morphologywith amyloidal nature as evident from SEM, FTIR and XRD experiments. The findings highlight that glycationinduced modification can be a possible approach for modulating the conformation of the silk protein which may berelevant in connection to clinical, biomedical or synthetic biology based applications.

Isolation and characterization of Lepidoptera specific Bacillus thuringiensis strains predominantly from north-eastern states of India.

Both, the tobacco caterpillar Spodoptera litura (Fabricius) and the cotton bollworm Helicoverpa armigera (Hübner), are serious polyphagous pests causing considerable loss to crops. Indiscriminate use of chemical pesticides for controlling them has rather resulted in their resistance development. Microbial pesticides, Bacillus thuringiensis in particular, play an important role in pest management. Here, we isolated Bacillus thuringiensis-like bacteria from the soil samples primarily collected from North East region of India along with some states viz., Haryana, Punjab, Maharashtra, West Bengal and Uttarakhand and studied their toxicity against the above two insect pests at 10 µg/g along with standard strain B. thuringiensis subsp. kurstaki HD-1 and at 1 µg/g Pseudomonas fluorescens based MVPII expressing Cry1Ac toxin and AUG-5. Isolates AUG-5 and GTG-7 proved toxic to more than 75% larvae on the 4th as well as 7th day of the treatment of the neonates of H. armigera. The AUG-5 isolate was also effective against S. litura. Ten effective isolates (AUG-5, GTG-4, GTG-7, GTG-9, GTG-42, GTG-64, GTG-70, GTG-3S, GTG-4S and GTG-6S) were characterized using biochemical and 16S rDNA analysis. Nearly, all the isolates tested positive for utilizing monosaccharides. All selected B. thuringiensis isolates showed resistance to ampicillin and co-trimoxazole except AUG-5 to co-trimoxazole. AUG-5 and GTG-7 were highly toxic to both insects, and possessed cry1, cry1A and cry2 genes. These isolates AUG-5 and GTG-7 also contained high Cry1Ac (104.8 and 88.32 ng/mg) and Cry2Ab (3792 and 1305.9 ng/mg), respectively in their spore-crystal complex. Both, AUG-5 and GTG-7 isolates, could be considered for further development as bioinsecticides. The present study has established the diversity and richness of B. thuringiensis-like isolates in soils collected from north-eastern region of India.

Insecticidal Activity of Ethyl Acetate Extracts from Culture Filtrates of Mangrove Fungal Endophytes

In the search for novel potent fungi-derived bioactive compounds for bioinsecticide applications, crude ethyl acetate culture filtrate extracts from 110 mangrove fungal endophytes were screened for their toxicity. Toxicity tests of all extracts against brine shrimp (Artemia salina) larvae were performed. The extracts with the highest toxicity were further examined for insecticidal activity against Spodoptera litura larvae and acetylcholinesterase (AChE) inhibition activity. The results showed that the extracts of five isolates exhibited the highest toxicity to brine shrimp at 50% lethal concentration (LC50) values of 7.45 to 10.24 ppm. These five fungal isolates that obtained from Rhizophora mucronata were identified based on sequence data analysis of the internal transcribed spacer region of rDNA as Aspergillus oryzae (strain BPPTCC 6036), Emericella nidulans (strains BPPTCC 6035 and BPPTCC 6038), A. tamarii (strain BPPTCC 6037), and A. versicolor (strain BPPTCC 6039). The mean percentage of S. litura larval mortality following topical application of the five extracts ranged from 16.7% to 43.3%. In the AChE inhibition assay, the inhibition rates of the five extracts ranged from 40.7% to 48.9%, while eserine (positive control) had an inhibition rate of 96.8%, at a concentration of 100 ppm. The extracts used were crude extracts, so their potential as sources of AChE inhibition compounds makes them likely candidates as neurotoxins. The high-performance liquid chromatography profiles of the five extracts differed, indicating variations in their chemical constituents. This study highlights the potential of culture filtrate ethyl acetate extracts of mangrove fungal endophytes as a source of new potential bioactive compounds for bioinsecticide applications.

Study on larvicidal activity of weed extracts against Spodoptera litura.

Larvicidal activity of various concentrations of crude aqueous leaf extracts of E. triplinerve were studied against S. litura following the treatment on fourth instar larvae. The results of the total percent mortality (from fourth instar to adult) demonstrated that the mortality rate progressively increased (41.42, 58.57, 69.80, 84.49, 86.77, 90.55, 91.30, 90.54) as the extract concentrations increased (1, 2, 3, 5, 7.5, 12.5, 15, 17.5%). The maximum of 92 % total mortality was observed at 15 % concentration of the extract. It was significant to note that the percent mortality observed from 5 % onwards was more than 85 %. Results of adult emergence inhibition indicated that the EI50 of E triplinerve was more effective at 4.07 %. Interestingly the calculated EI90 value was 14.10 %. This is an ideal eco-friendly approach for the control of agricultural pest, S. litura.

Effect of PONNEEM# on Spodoptera litura (Fab.) and its compatibility with Trichogramma chilonis Ishii

Antifeedant and growth regulating activities of PONNEEM, an oil formulation containing neem and pungam (karanj) oils were evaluated along with individual neem and karanj oils and Nimbicidine, a commercial neem-based pesticide against fourth instar larvae of Spodoptera litura (Fab.). Among all the treatments, PONNEEM recorded the maximum antifeedant activity (88.6%) at 0.6%. Neem and karanj individual treatments as well as PONNEEM extended larval duration compared to control. Pupal weight and fecundity were significantly reduced and pupal period was greatly increased by PONNEEM treatment compared to other treatments. The PONNEEM was found to be compatible with Trichogramma chilonis Ishii, an egg parasitoid of many lepidopteran pests, at 0.15, 0.3 and 0.5% concentrations. PONNEEM did not affect the parasitoid emergence significantly at 0.3% concentration compared to control. PONNEEM also showed growth disruption activity against fourth instar larvae.

Biological activities of Solanum pseudocapsicum (Solanaceae) against cotton bollworm, Helicoverpa armigera Hübner and armyworm, Spodoptera litura Fabricius (Lepidotera: Noctuidae)

<p><b>OBJECTIVE</b>To evaluate the antifeedant, insecticidal and growth inhibition activities of Solanum pseudocapsicum (S. pseudocapsicum) seed extracts against Spodoptera litura (S. litura) and Helicoverpa armigera (H. armigera).</p><p><b>METHODS</b>Hexane, diethyl ether, dichloromethane and ethyl acetate seed extracts were prepared and tested for antifeedant, insecticidal and growth inhibitory activities against fourth instar larvae of S. litura and H. armigera.</p><p><b>RESULTS</b>Ethyl acetate extract showed promising antifeedant and insecticidal activities against S. litura and H. armigera. Percentage of deformed larvae, pupae and adults were maximum in treatment of ethyl acetate extract. Percentage of successful adult emergence was deteriorated by seeds on extract treated larvae.</p><p><b>CONCLUSIONS</b>Ethyl acetate extracts of S. pseudocapsicum, showed higher efficiency of antifeedant, insecticidal and growth inhibition activities. Hence, it can be used to controll agricultural insect pests, S. litura and H. armigera.</p>

Ovicidal activity of Atalantia monophylla (L) Correa against Spodoptera litura Fab. (Lepidoptera: Noctuidae)

<p><b>OBJECTIVE</b>To evaluate the efficacy of Atalantia monophylla (A. monophylla) leaf in different solvent crude extracts and fractions against eggs of Spodoptera litura (S. litura).</p><p><b>METHODS</b>Hexane, ethyl acetate and chloroform solvent extracts of A. monophylla leaf and 12 fractions from hexane extract were screened at 5.0%, 2.5%, 1.0% and 0.5% for crude extracts and 1 000, 500, 250 and 125 mg/kg for fractions against the eggs of S. litura for the ovicidal activity. LC50 and LC90 were calculated using probit analysis.</p><p><b>RESULTS</b>Hexane crude extract showed maximum ovicidal activity of 61.94% at 5.0% concentration with a correlation value of r (2)=0.81, and least LC50 value of 3.06%. Hexane extract was fractionated using silica gel column chromatography and 12 fractions were obtained. Fraction 9 was active which showed maximum ovicidal activity of 75.61% at 1 000 mg/kg with the LC50 value of 318.65 mg/kg and LC90 value of 1 473.31 mg/kg. In linear regression analysis, significant and high correlation (r (2)=0.81%) was seen between concentration and ovicidal activity of hexane crude extracts and its active fraction.</p><p><b>CONCLUSIONS</b>As per our knowledge, this is the first report for ovicidal activity of A. monophylla against S. litura, A. monophylla could be used for the management of S. litura and other insect pests.</p>

Evaluation of Strains of Metarhizium anisopliae and Beauveria bassiana against Spodoptera litura on the Basis of Their Virulence, Germination Rate, Conidia Production, Radial Growth and Enzyme Activity

Ten strains of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were evaluated to find the most effective strain for optimization studies. The first criterion tested for strain selection was the mortality (> 50%) of Spodoptera litura larvae after inoculation of the fungus for 4 days. Results on several bioassays revealed that B. bassiana BNBCRC showed the most virulence on mortality S. litura larvae (80% mortality). B. bassiana BNBCRC also showed the highest germination rate (72.22%). However, its conidia yield (7.2 x 10(8) conidia/mL) was lower than those of B. bassiana B 14841 (8.3 x 10(8) conidia/mL) and M. anisopliae M6 (8.2 x 10(8) conidia/mL). The highest accumulative radial growth was obtained from the strain B14841 (37.10 mm/day) while the strain BNBCRC showed moderate radial growth (24.40 mm/day). M. anisopliae M6 possessed the highest protease activity (145.00 mU/mL) while M. anisopliae M8 possessed the highest chitinase activity (20.00 mU/mL) during 96~144 hr cultivation. Amongst these criteria, selection based on virulence and germination rate lead to the selection of B. bassiana BNBCRC. B. bassiana B14841 would be selected if based on growth rate while M. anisopliae M6 and M8 possessed the highest enzyme activities.

Biological activities of Solanum pseudocapsicum (Solanaceae) against cotton bollworm, Helicoverpa armigera Hübner and armyworm, Spodoptera litura Fabricius (Lepidotera:Noctuidae)

Objective:To evaluate the antifeedant, insecticidal and growth inhibition activities of Solanum pseudocapsicum (S. pseudocapsicum) seed extracts against Spodoptera litura (S. litura) and Helicoverpa armigera (H. armigera). Methods:Hexane, diethyl ether, dichloromethane and ethyl acetate seed extracts were prepared and tested for antifeedant, insecticidal and growth inhibitory activities against fourth instar larvae of S. litura and H. armigera. Results:Ethyl acetate extract showed promising antifeedant and insecticidal activities against S. litura and H. armigera. Percentage of deformed larvae, pupae and adults were maximum in treatment of ethyl acetate extract. Percentage of successful adult emergence was deteriorated by seeds on extract treated larvae. Conclusions: Ethyl acetate extracts of S. pseudocapsicum, showed higher efficiency of antifeedant, insecticidal and growth inhibition activities. Hence, it can be used to controll agricultural insect pests, S. litura and H. armigera.

Ovicidal activity of Atalantia monophylla (L) Correa against Spodoptera litura Fab. (Lepidoptera:Noctuidae)

Objective: To evaluate the efficacy of Atalantia monophylla (A. monophylla) leaf in different solvent crude extracts and fractions against eggs of Spodoptera litura (S. litura). Methods:Hexane, ethyl acetate and chloroform solvent extracts of A. monophylla leaf and 12 fractions from hexane extract were screened at 5.0%, 2.5%, 1.0%and 0.5%for crude extracts and 1 000, 500, 250 and 125 mg/kg for fractions against the eggs of S. litura for the ovicidal activity. LC50 and LC90 were calculated using probit analysis. Results:Hexane crude extract showed maximum ovicidal activity of 61.94%at 5.0%concentration with a correlation value of r2=0.81, and least LC50 value of 3.06%. Hexane extract was fractionated using silica gel column chromatography and 12 fractions were obtained. Fraction 9 was active which showed maximum ovicidal activity of 75.61%at 1 000 mg/kg with the LC50 value of 318.65 mg/kg and LC90 value of 1 473.31 mg/kg. In linear regression analysis, significant and high correlation (r2=0.81%) was seen between concentration and ovicidal activity of hexane crude extracts and its active fraction. Conclusions:As per our knowledge, this is the first report for ovicidal activity of A. monophylla against S. litura, A. monophylla could be used for the management of S. litura and other insect pests.

Ovicidal activity of botanical oil formulations against Helicoverpa armigera Hubner and Spodoptera litura Fabricius (Lepidoptera：Noctuidae)

Objective: To evaluate the ovicidal activity of different botanical oil formulations againstHelicoverpa armigera and Spodoptera litura. Methods: Different botanical oils were formulated with different ratio to evaluate the ovicidal activity against H. armigera and S. litura at 5, 10, 15 and 20μl/L concentrations. Results: All the oil formulations showed the ovicidal activity against H. armigera and S. litura. The maximum ovicidal actvity of 76.74 and 69.36% was noticed at 20μl/L concentration in formulation 3 PONNEEM. Formulation 4 Pongam oil showed lower ovicidal activity of 31.34 and 24.76% against H. armigera and S. litura respectively. Among the formulations, PONNEEM exhibited statistically superior ovicidal activity against both insect pests. Conclusions: the present study clearly showed PONNEEM as a pontenial biopesticide to control the egg stage of economically important pests of H. armigera and S. litura. This is the first report for the ovicidal activity of PONNEEM against these two insect pests.

Recombinant expression and refolding of the c-type lysozyme from Spodoptera litura in E. coli

The chicken-type lysozyme of the insect Spodoptera litura (SLLyz) is a polypeptide of 121 amino acids containing four disulfide bridges and 17 rare codons and participates in innate defense as an anti-bacterial enzyme. The recombinant S. litura lysozyme (rSLLyz) expressed as a C-terminal fusion protein with glutathione S-transferase (GST) in Rosetta(DE3) Singles. The protein was produced as an inclusion body which was solubilized in 8 M urea, renatured by on-column refolding, and purified by reversed-phase chromatography to 95 percent purity. The purified rSLLyz demonstrated antibacterial activity against B. megaterium confirmed by inhibition zone assay. The overexpression and refolding strategy described in this study will provide a reliable technique for maximizing production and purification of proteins expressed as inclusion bodies in E. coli.