Insecticidal Activity of the Granulosis Virus in Combination with Neem Products and Talc Powder Against the Potato Tuberworm Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)

The potato tuberworm Phthorimaea operculella (Zeller) is an important agricultural pest that causes significant economic losses to potato growers worldwide. The addition of an effective method of biological control for the potato tuberworm is greatly needed, and is currently unavailable in Brazil. The granulosis virus (Baculoviridae) is a promising biological control agent to protect post-harvest potatoes and in storage from the potato tuberworm. However, the control measure must be economically feasible. Liquid suspensions of a granulosis virus applied alone or in mixture with two commercial neem oil-based products (DalNeem (TM) and NeemAzal (TM)), and a dry powder formulation of viral granules were evaluated for control of potato tuberworm larvae by treating potato tubers under laboratory conditions. High larval mortality (86.7%) was achieved when DalNeem and virus were applied together at 4 mg of azadirachtin/L and 10(4) occlusion bodies (OBs)/mL, respectively. This combination resulted in a parts per thousand yen50% efficacy in relation to their counterparts alone. Conversely, NeemAzal did not enhance virus effectiveness against larvae of the potato tuberworm. The talc-based virus formulation was used for dusting seed tubers at different concentrations and resulted in 100% larval mortality at 5 x 10(8) OBs/g. Formulated and unformulated virus provided 50% mortality at 166 OBs/g and at 5.0 x 10(5) OBs/mL, respectively. As a result, talc-based virus formulation had a better control efficiency on potato tuberworm than the aqueous virus suspension. The granulosis virus combined with DalNeem at low rates or formulated with talc powder is a viable option to control the potato tuberworm under storage conditions.

Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II

Griffonia simplicifolia leaf lectin II (GSII), a plant defense protein against certain insects, consists of an N-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid, Callosobruchus maculatus (F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.

Involvement of loop 5 lysine residues and the N-terminal β-hairpin of the ribotoxin hirsutellin A on its insecticidal activity

Ribotoxins are cytotoxic members of the family of fungal extracellular ribonucleases best represented by RNase T1. They share a high degree of sequence identity and a common structural fold, including the geometric arrangement of their active sites. However, ribotoxins are larger,with a well-defined N-terminal β-hairpin, and display longer and positively charged unstructured loops. These structural differences account for their cytotoxic properties.Unexpectedly, the discovery of hirsutellin A (HtA), a ribotoxin produced by the invertebrate pathogen Hirsutella thompsonii, showed how it was possible to accommodate these features into a shorter amino acid sequence. Examination of HtA N-terminal β-hairpin reveals differences in terms of length, charge, and spatial distribution. Consequently,four different HtA mutants were prepared and characterized. One of them was the result of deleting this hairpin [Δ(8-15)] while the other three affected single Lys residues in its close spatial proximity (K115E, K118E, and K123E). The results obtained support the general conclusion that HtA active site would show a high degree of plasticity,being able to accommodate electrostatic and structural changes not suitable for the other previously known larger ribotoxins, as the variants described here only presented small differences in terms of ribonucleolytic activity and cytotoxicity against cultured insect cells.

Isolation, solution structure, and insecticidal activity of Kalata B2, a circular protein with a twist: Do Mobius strips exist in nature?

A large number of macrocyclic miniproteins with diverse biological activities have been isolated from the Rubiaceae, Violaceae, and Cucurbitaceae plant families in recent years. Here we report the three-dimensional structure determined using H-1 NMR spectroscopy and demonstrate potent insecticidal activity for one of these peptides, kalata B2. This peptide is one of the major components of an extract from the leaves of the plant Oldenlandia affinis. The structure consists of a distorted triple-stranded beta-sheet and a cystine knot arrangement of the disulfide bonds and is similar to those described for other members of the cyclotide family. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals that they can be separated into two subfamilies, one of which contains a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-peptidyl-proline bond and may conceptually be regarded as a molecular Mobius strip. Kalata B2 is the second putative member of the Mobius cyclotide family to be structurally characterized and has a cis-peptidyl-proline bond, thus validating the suggested name for this subfamily of cyclotides. The observation that kalata B2 inhibits the growth and development of Helicoverpa armigera larvae suggests a role for the cyclotides in plant defense. A comparison of the sequences and structures of kalata B1 and B2 provides insight into the biological activity of these peptides.

Insecticidal activity of a protein extracted from bulbs of Phycella australis Ravenna against the aphids Acyrthosiphon pisum Harris and Myzus persicae Sulzer

Aphids cause significant losses in many agricultural crops and in many cases cause repeated insecticide sprays, which increase the risk of resistance. Therefore, other alternatives are needed to control them. The toxic, antireproductive, and feeding deterrent effects of a mannosebinding lectin isolated from bulbs of Phycella australis Ravenna (Amaryllidaceae), named Phycella australis agglutinin (PAA) was assayed on nymphs of the aphids Acyrthosiphon pisum Harris and Myzus persicae Sulzer fed with an artificial diet. After 72 h of PAA exposure, lethal concentration (LC50) values were 109 and 313 μg mL-1 for A. pisum and M. persicae, respectively, while LC90 values were 248 and 634 μg mL-1. Sub-lethal concentrations of PAA significantly reduced the aphid fecundity at a concentration of 80 μg mL-1. Only a total of 5.7 descendants per female were recorded for A. pisum (32% control progeny) and 12.4 for M. persicae (39% control progeny). Acyrthosiphon pisum was strongly deterred by PAA under choice conditions, as after 72 h exposed to 80 μg PAA mL-1 of diet, the feeding deterrent index was 0.91 for A. pisum and only 0.38 for M. persicae. In conclusion, the mannosebinding lectin isolated from bulbs of P. australis showed acute and chronical insecticidal activity against the pea and green peach aphids.

Oligomerization of Cry9Aa in solution without receptor binding, is not related with insecticidal activity

Background: Bacillus thuringiensis Cry toxins bind with different insect midgut proteins leading to toxin oligomerization, membrane insertion and pore formation. However, different Cry toxins had been shown to readily form high molecular weight oligomers or aggregates in solution in the absence of receptor interaction. The role of Cry oligomers formed in solution remains uncertain. The Cry9A proteins show high toxicity against different Lepidoptera, and no-cross resistance with Cry1A. Results: Cry9Aa655 protein formed oligomers easily in solution mediated by disulfide bonds, according to SDS-PAGE analysis under non-reducing and reducing conditions. However, oligomerization is not observed if Cry9Aa655 is activated with trypsin, suggesting that cysteine residues, C14 and C16, located in the N-terminal end that is processed during activation participate in this oligomerization. To determine the role of these residues on oligomerization and in toxicity single and double alanine substitution were constructed. In contrast to single C14A and C16A mutants, the double C14A–C16A mutant did not form oligomers in solution. Toxicity assays against Plutella xylostella showed that the C14A–C16A mutant had a similar insecticidal activity as the Cry9Aa655 protein indicating the oligomers of Cry9Aa formed in solution in the absence of receptor binding are not related with toxicity. Conclusions: The aggregation of Cry9Aa655 polypeptides was mediated by disulfide bonds. Cry9Aa655 C14 and C16C are involved in oligomerization in solution. These aggregate forms are not related to the mode of action of Cry9Aa leading to toxicity.

Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops. (C) 2010 Elsevier Inc. All rights reserved.

Trypanocidal Activity of Limonoids and Triterpenes from Cedrela fissilis

Chagas` disease is an illness that affects millions of people in Central and South America, The search for both a prophylactic drug to be added to human blood as well as a safe and reliable therapeutic drug are greatly needed to control such disease. Herein, we report the trypanocidal activity of 15 crude extracts and 14 Compounds (limonoids and triterpenes) as well as the isolation of 25 known compounds (6 limonoids, 12 triterpenes, 1 sesquiterpene, 5 steroids, and 1 flavonoid) from Cedrela fissilis. The present study shows that this plant is a Promising Source of active compounds for the control of Chagas` disease. The inhibitory activity found for odoratol indicates that it is potentially useful as an alternative for the chemoprophylactic gentian violet.

Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge

We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.

Functional significance of the beta-hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a

omega -Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels. In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions. First, we constructed a Hairpinless mutant in which the C-terminal beta -hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial hits from the alanine scan. This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity. We propose that this surface of the beta -hairpin is a key site for interaction of the toxin with insect calcium channels.

Promise for plant pest control: root-associated pseudomonads with insecticidal activities.

Insects are an important and probably the most challenging pest to control in agriculture, in particular when they feed on belowground parts of plants. The application of synthetic pesticides is problematic owing to side effects on the environment, concerns for public health and the rapid development of resistance. Entomopathogenic bacteria, notably Bacillus thuringiensis and Photorhabdus/Xenorhabdus species, are promising alternatives to chemical insecticides, for they are able to efficiently kill insects and are considered to be environmentally sound and harmless to mammals. However, they have the handicap of showing limited environmental persistence or of depending on a nematode vector for insect infection. Intriguingly, certain strains of plant root-colonizing Pseudomonas bacteria display insect pathogenicity and thus could be formulated to extend the present range of bioinsecticides for protection of plants against root-feeding insects. These entomopathogenic pseudomonads belong to a group of plant-beneficial rhizobacteria that have the remarkable ability to suppress soil-borne plant pathogens, promote plant growth, and induce systemic plant defenses. Here we review for the first time the current knowledge about the occurrence and the molecular basis of insecticidal activity in pseudomonads with an emphasis on plant-beneficial and prominent pathogenic species. We discuss how this fascinating Pseudomonas trait may be exploited for novel root-based approaches to insect control in an integrated pest management framework.

Activity and Residual Effect of Two Formulations of Lambdacyhalothrin Sprayed on Palm Leaves to Rhodnius prolixus

The insecticidal activity and residual effect of two formulations of lambdacyhalothrin were evaluated with Rhodnius prolixus;laboratory and field tests were conducted in the State of Chiapas, Mexico. The results indicate that the lethal concentrations of the active ingredient of SC (LC50 = 2.37 and LC90 = 8.5 mg, a.i./m²) were 4-8 times than those with the insecticide WP applied on R. prolixus bugs in palm leaves, a common building material for thatched roofs. Other investigators in South America recommended applying 30 mg a.i./m² in porous materials; we obtained that the products WP and SC were 3.5 and 16 times more effective on palm leaves. Regarding the evaluation of the residual effects in field spraying, there was up to 15 months persistence after the application of WP in two doses (8.6 mg a.i./m² and 3.752 mg a.i./m²) with SC. We consider R. prolixus highly susceptible to the employed pyrethroids; they could be used to control this vector in the state of Chiapas, Mexico.

Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus.

BACKGROUND: Root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. Some of these bacteria produce an insecticidal toxin (Fit) suggesting that they may exploit insect hosts as a secondary niche. However, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling. RESULTS: Screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the Fit toxin revealed that Fit is highly indicative of insecticidal activity and predicts that Pseudomonas protegens and P. chlororaphis are exclusive Fit producers. A comparative evolutionary analysis of Fit toxin-producing Pseudomonas including the insect-pathogenic bacteria Photorhabdus and Xenorhadus, which produce the Fit related Mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in GC base composition. The patchy distribution and phylogenetic incongruence of fit genes indicate that the Fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique Pseudomonas-specific insect toxin cluster. CONCLUSIONS: Our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the Mcf/Fit toxin highlighting the dynamic nature of insect toxin evolution.

Activity of Ricinus communis (Euphorbiaceae) and ricinine against the leaf-cutting ant Atta sexdens rubropilosa (Hymenoptera : Formicidae) and the symbiotic fungus Leucoagaricus gongylophorus

The focus of this study was the identification of compounds from plant extracts for use in crop protection. This paper reports on the toxic activity of fractions of leaf extracts of Ricinus communis L (Euphorbiaceae) and isolated active compounds in the leaf-cutting ant Atta sexdens rubropilosa Forel and its symbiotic fungus Leucoagaricus gongylophorus (Singer) Moller. The main compounds responsible for activity against the fungus and ant in leaf extracts of R communis were found to be fatty acids for the former and ricinine for the ants. (C) 2004 Society of Chemical Industry.

Biological activity of astilbin from Dimorphandra mollis against Anticarsia gemmatalis and Spodoptera frugiperda

Astilbin was isolated in high yield from Dimorphandra mollis, and its insecticidal and growth inhibiting activity by stomach ingestion were evaluated against Anticarsia gemmatalis and Spodoptera frugiperda. The insecticidal activity of astilbin, the weight reduction of the larval phase and the prolongation of the larval and pupal phases were verified for both species. Astilbin was identified on the base of its NMR, MS and physical data. (C) 2002 Society of Chemical Industry.