Promising active bioinsecticides produced by Bacillus thuringiensis strain BLB427

Isolation and characterization of a new Bacillus thuringiensis strain with a promising toxicity against Lepidopteran pests

Conjugation of δ‐endotoxin from Bacillus thuringiensis with abamectin, a toxin of Streptomyces avermitilis, was carried out to form a new type of biocide, GCSC‐BtA based on “Germany‐China Scientific Cooperation” research, for the control of agricultural insect pests. The strategy for biochemical linkage was designed by conjugating an amino group in B.t. protoxin with a carboxyl group in carboxylated abamectin under the treatment of conjugator EDC [1‐Ethyl‐3‐(3‐dimethylaminopropyl carbodiimide Hydrochloride)]. The formation of B.t. protoxin was processed by solubilizing B.t. crystal in 25 mM dithiothreitol (DTT) at 37 °C for 2 h. The carboxylated abamectin was formed by carboxylating the NaH‐activated abamectin with 10 mg/ml butyric anhydride at 111 °C in a water‐circumfluent condensation device for 2 h. The conjugating reaction, consisting of 5 mg/ml B.t. protoxin, 10 mg/ml carboxylated abamectin and 19.17 mg/ml EDC, was successfully conducted at room temperature for 24 h. Significant differences were found between pure abamectin, carboxylated abamectin and the conjugated BtA by means of UV‐photo absorptions recorded at wavelengths 354, 438, 518, 600 nm (P < 0.01). LT50 of the conjugated GCSC‐BtA to the 3rd instar larvae of Plutella xylostella (L.) (Lep., Plutellidae) was 35.27 μg a.i./ml, about 62 % and 76 % of that caused by the B.t. protoxin and the caxboxylated abamectin, respectively. The conjugated GCSC‐BtA caused 87.14 % mortalities in larvae of P. xylostella, 93.75 % in adult Myzus persicae (Sulzer) (Hom., Aphididae) and 89.33 % in adult Phyllotreta vittata Fabricius (Col., Chrysomelidae) as compared to 48.33 % by the B.t. crystal only in P. xylostella. The symptoms caused by conjugated GCSC‐BtA in the 3rd instar of P. xylostella were black color in the head part and white‐yellow in the abdomen of dead larvae, which differed from the black color or the white‐yellow all along the body caused by either the B.t. crystal or the abamectin, respectively. It was concluded that the conjugated GCSC‐BtA biocide had a broader host spectrum and a faster killing speed than either the B.t. crystal or abamectin alone for the control of agricultural pests.

Different microbial insect pathogens have been used as insecticides for the control of different insect pests in agriculture and also for the control of insects that are vectors of important human diseases. These microbial pathogens include bacteria, viruses and fungi. These microbes differ in their insect specificity and mode of action. Nevertheless, insecticides based on these microbial insect pathogens have had a limited use in comparison to chemical insecticides. In this chapter we will revise the potential use of some of these microbial pathogens highlighting their mode of action, insect specificity, risk assessments for the environment and also key application practices to assure the development of more healthy insect control products. Bacterial pathogens rely on different virulence factors to invade their hosts. Among these, bacterial toxins are by far the most important virulence factors. Some δ-endotoxins produced by Bacillus thuringensis, have been expressed in transgenic crops resulting in the reduction of the use of chemical insecticides. Other insecticidal toxins from other bacterial species are likely to provide additional tool for insect control in agriculture. Although different viruses and fungi have the potential for use as insect control, their wide use has been limited by their narrow spectrum of action, application practices and in some cases low susceptibility by older larval instars. However, the genetic manipulation of their genomes by the introduction of insect toxins or catabolic enzymes has been shown to significantly improve their insecticidal activity and their potential for wider use as insect control agents.

Cry proteins, produced by Bacillus thuringiensis (Bt), are widely used for the control of insect pests in agriculture as spray products or expressed in transgenic crops, such as maize and cotton. Little was known regarding the mechanism of action of these toxins when the first commercial Bt product was introduced fifty years ago. However, research on the mechanism of action over the last two decades has enhanced our knowledge of toxin interaction with membrane receptors and their effects in insect midgut cells. All this information allowed for the rational design of improved toxins with higher toxicity or toxins that overcome insect resistance, which could compromise Bt use and effectiveness in the field. In this review we discuss and evaluate the different models of the mode of action of Cry toxins, including a discussion about the role of various receptors in toxin action.

The mechanism of damage to the posterior silk gland by trace amounts of acetamiprid in the silkworm, Bombyx mori

The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.

Bacillus thuringiensis (Bt) produces vegetative insecticidal proteins (Vip) during its vegetative growth stage. Vip3 proteins have a significant role in insecticidal activity of this bacterium. Each new bacterial isolate may encode Vip with different significance. The Vip protein coding by vip genes of two Bt strains (BnBt and MnD) with high insecticidal activity was characterized in this study. Polymerase chain reaction (PCR)-based screening for Vip genes of these Bt isolates expressed the detected Vip gene and tested the protein for insecticidal activity against the cotton leafworm, Spodoptera littoralis larvae. As a result of the screening tests, Vip3 genes were determined in MnD and BnBt isolates. Vip3 genes of both isolates were expressed and confirmed by 90 kDa proteins. Partially purified and trypsin-activated protein samples of BnBt and MnD isolates were tested against the second instar larvae of S. littoralis. The results showed that the highest insecticidal activity of the Vip3 proteins of BnBt and MnD was 86.66% and 83.33% mortality in 10 days, respectively. The median lethal concentrations (LC50) of BnBt and MnD were determined as 41.860 and 55.154 ng/μl, respectively. The results suggest that Vip3 protein is effective for preventing resistance in various insect-pest species. The expressed proteins may be utilized as a biopesticide against nocuous insects.

Nanoparticles and plants: A focus on analytical characterization techniques

Pesticides are one of the most alarming toxic substances that are deliberately added to our environment. Food and agricultural organization (FAO) has defined the term pesticide as: Any substance or mixture of substances intended for preventing, destroying or controlling any pest including vectors of human or animal disease, unwanted species of plants or animals causing harm during or otherwise interfering with the production, processing, storage, transport or marketing of food, agricultural commodities, wood and wood products or animal feedstuffs or substances which may be administered to animals for control of insects, arachnids or other pests in or on their bodies [1]. But it is a matter of concern that along with the pests, they prove harmful to many other living beings on this planet as well as human beings including newborns [2]. CARBARYL, a synthetic 1-napthyl-N-methyl carbamate is being used extensively or its broad-spectrum activity in commercial agriculture, poultry, livestock, home and garden pest control. It was the most frequently detected carbamate in juice samples studied [3]. Thorough scanning reveals that there is dearth of literature regarding the histopathological changes in liver associated with administration of carbaryl which has prompted us to initiate this study as liver plays an important role in the first pass metabolism of carbaryl. The present study was conducted on 40 albino rats. The adult albino rats (150-200 gm) were injected with 200 mg/ kg body weight of carbaryl intrapeitoneally, five days a week for 30 days. Controls were maintained. There was a significant increase in weight of the experimental rats recorded before the onset of the experiment & prior to their sacrifice (p<0.001) as compared to the controls. The rats were sacrificed within 24 hours of the last injection. The blocks of the liver were prepared for section cutting with a microtome by paraffin wax embedding method. Sections of 5-7 µm thickness were cut and stained with Haematoxylin and Eosin stain. In the study, the histomorphological changes, in the liver of Carbaryl treated rats was significantly different from that of the normal and the control rats. The disheveled pattern of the one cell thick orderly arrangement of hepatocytic cords, evidence of increased cellular metabolism co-existent with ballooning degeneration, councilman bodies, inflammatory infiltrate around the portal triads along with the dilatation of the blood vessels and the bile canaliculi were seen as signs of degeneration. B enucleated hepatocytes were suggestive of regenerative attempts by the degenerative cells. These findings are highly conclusive of toxic hepatitis induced by an insecticide, Carbaryl.

Bacillus thuringiensis (Bt) is a bacterial genus known mainly for its capability to synthesize, in addition to spores, a parasporal body or crystal (δ––endotoxin), comprised of proteins which are toxic to pest insects, i.e., Coleoptera, Diptera and Lepidoptera, and other orders. As well as plant pathogenic nematode and many other applications as endophytic plant growth promoting bacteria, or cleaning enviornmental form some chemicals polluting agents, and even in human medicine for cancer prevention. The Bt coexists in a close relationship with insects to which eventually infects and poisons. It is likely that these unfavorable changes occurred during the evolutionary pathway in the interactions Bacillus–insect, which resulted in the present biochemical diversity between these species. The goal of this minireview is to show role of Bt in comparation with other genus Bacillus and explain why Bt is of interest for biological control of pests in conventional, protected and organic agriculture regarding other biotechnological applications to fully exploit the beneficial potential of Bt

A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.

Climate change affects the physiology of insects by accelerating their metabolism, growth, and reproduction, which increases their density, and ultimately leads to the consumption and infestation of crops damaged, this action, contributes to economic losses, especially in countries where agriculture plays an important role in their economic sector. This chapter aims to identify how the climatic parameters, in particular the rise in temperature that promotes the development of its insect pests, also causes the action of expanding their distribution, and further reduces biological control, especially natural enemies. And also this chapter shows how insects interact with crops to cause agricultural infestations and damage. The control of agricultural pests can only be established through in-depth studies on the impact of climate change on the interaction between agricultural pests and crops and the contribution of strategies that incorporate the environment and physical resources, and social and financial benefits for farmers. Moreover, the application of adaptation practices, such as improved pest management, monitoring of climate and pest populations, and development of integrated models for predicting climate change, agriculture, and pests, are also considered relevant and effective solutions for the pest control.

Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could, therefore, restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent virtual screening to produce nano-/picomolar boronic acid inhibitors of the carboxylesterase αE7 from the agricultural pest Lucilia cuprina as well as a common Gly137Asp αE7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase and low to no toxicity in human cells and in mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos, against the common pest, the peach-potato aphid (Myzus persicae). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy.

The coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) was first reported infecting Costa Rican coffee plantations in the year 2000. Due to the impact that this plague has in the economy of the country, we were interested in seeking new alternatives for the biological control of H. hampei, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 202 B. thuringiensis isolates obtained from Costa Rican coffee plantations infested with H. hampei were analyzed through crystal morphology of the crystal inclusions and SDS-PAGE of 6-endotoxins, while 105 strains were further evaluated by PCR for the presence cry, cyt and vip genes. Most of the Bt strains showed diverse crystal morphologies: pleomorphic (35%), oval (37%), bipyramidal (3%), bipyramidal and oval (12%), bipyramidal, oval and pleomorphic (10%) and bipyramidal, oval and cubic (3%). The SDS-PAGE analyses of the crystal preparations showed five strains with delta-endotoxin from 20 to 40 kDa, six from 40 to 50 kDa, seven from 50 to 60 kDa, 19 from 60 to 70 kDa, 29 from 70 to 100 kDa and 39 from 100-145 kDa. PCR analyses demonstrated that the collection showed diverse cry genes profiles having several genes per strain: 78 strains contained the vip3 gene, 82 the cry2 gene, 45 the cry1 and 29 strains harbored cry3-cry7 genes. A total of 13 strains did not amplified with any of the cry primers used: cry1, cry2, cry3-7, cry5, cry11, cry12 and cry14. Forty-three different genetic profiles were found, mainly due to the combination of cry1A genes with other cry and vip genes. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against H. hampei and other insect pests of agricultural importance.

Selective control of mite and collembolan pests of pastures and grain crops in Australia