Interactions between Bacillus thuringiensis and selected plant extracts for sustainable management of Phthorimaea absoluta.

Phthorimaea absoluta is a global constraint to tomato production and can cause up to 100% yield loss. Farmers heavily rely on synthetic pesticides to manage this pest. However, these pesticides are detrimental to human, animal, and environmental health. Therefore, exploring eco-friendly, sustainable Integrated Pest Management approaches, including biopesticides as potential alternatives, is of paramount importance. In this context, the present study (i) evaluated the efficacy of 10 Bacillus thuringiensis isolates, neem, garlic, and fenugreek; (ii) assessed the interactions between the most potent plant extracts and B. thuringiensis isolates, and (iii) evaluated the gut microbial diversity due to the treatments for the development of novel formulations against P. absoluta. Neem recorded the highest mortality of 93.79 ± 3.12% with an LT50 value of 1.21 ± 0.24 days, Bt HD263 induced 91.3 ± 3.68% mortality with LT50 of 2.63 ± 0.11 days, compared to both Bt 43 and fenugreek that caused < 50% mortality. Larval mortality was further enhanced to 99 ± 1.04% when Bt HD263 and neem were combined. Furthermore, the microbiome analyses showed that Klebsiella, Escherichia and Enterobacter had the highest abundance in all treatments with Klebsiella being the most abundant. In addition, a shift in the abundance of the bacterial genera due to the treatments was observed. Our findings showed that neem, garlic, and Bt HD263 could effectively control P. absoluta and be integrated into IPM programs after validation by field efficacy trials.

Entomopathogenic fungi based microbial insecticides and their physiological and biochemical effects on Spodoptera frugiperda (J.E. Smith).

Background: 'The fall armyworm, Spodoptera frugiperda', represents a significant threat to maize production, a major staple crop in Asian countries. Methods: In pursuit of more effective control of this insect pest, our study assessed the physiological and biochemical effects of the entomopathogenic fungus Metarhizium anisopliae against the larvae of S. frugiperda. Results: Results revealed that, following nine days of treatment, a high concentration of conidia (1.5x107 conidia/mL-1) was toxic to all stages of larvae (second to fifth instar), resulting in 97% mortality of the second instar, 89% mortality of the third instar, 77% mortality of the fourth instar, and 72% mortality of fifth instar. All larval instars were found to have dose-dependent mortality effects. Treated S. frugiperda larvae further displayed significant physiological, morphological, and behavioral changes. Here, treated larvae displayed significantly lower levels of acetylcholinesterase, α-carboxylesterase, and ß-carboxylesterase enzyme activity when compared to control groups. Treated larvae underwent an outward morphological change as the result of a decrease in the exterior cuticle of the anal papillae and a demelanization of the interior cuticle. Treated larvae also exhibited abnormal feeding behaviors as a consequence of the negative impact of conidia treatment on the neuromuscular system. Investigation into the effect of M. anisopliae on the non-target organism, the earthworm Eudrilus eugeniae, revealed that M. anisopliae conidia did not produce significant pathogenicity following three days of treatment. Furthermore, histological analysis revealed no significant effect of the entomopathogenic fungi on the gut tissue of the non-target organism. Conclusion: This study highlights the potential of M. anisopliae in the control of S. frugiperda.

Susceptibility of Hyphantria cunea larvae to Beauveria bassiana under Cd Stress: An integrated study of innate immunity and energy metabolism.

Biological control is widely used for integrated pest management. However, there are many abiotic factors that can affect the biocontrol efficiency. In this study, we investigated the susceptibility of Hyphantria cunea larvae to Beauveria bassiana under Cd stress, and the corresponding mechanism was analyzed around innate immunity and energy metabolism. The results showed that mortality of H. cunea larvae treated with Cd and B. bassiana was significantly higher than those treated with B. bassiana alone, and the combined lethal effect exhibited a synergistic effect. Compared with the single fungal treatment group, the total hemocyte count in the combined Cd and fungal treatment group decreased significantly, accompanied by a decrease in phagocytosis, encapsulation, and melanization activity. The expression levels of three phagocytosis-related genes, one encapsulation-promoting gene, and one melanization-regulating gene were significantly lower in the combined treatment group than those in the single fungal treatment group. Furthermore, pathogen recognition ability, signal transduction level, and immune effector expression level were weaker in the combined treatment group than those in the single fungal treatment group. The expression levels of 14 key metabolites and 7 key regulatory genes in glycolysis and tricarboxylic acid cycle pathways were significantly lower in the combined treatment group than those in the single fungal treatment group. Taken together, the weakness of innate immunity and energy metabolism in response to pathogen infection resulted in an increased susceptibility of H. cunea larvae to B. bassiana under Cd pre-exposure. Microbial insecticide is a preferred strategy for pest control in heavy metal-polluted areas. AVAILABILITY OF DATA AND MATERIAL: All the data that support the findings of this study are available in the manuscript.

First report on the enzymatic and immune response of Metarhizium majus bag formulated conidia against Spodoptera frugiperda: An ecofriendly microbial insecticide.

Entomopathogenic fungi from microbial sources are a powerful tool for combating insecticide resistance in insect pests. The purpose of the current study was to isolate, identify, and evaluate bag-formulated entomopathogenic fungal conidial virulence against insect pests. We further investigated the enzymatic responses induced by the entomopathogenic fungi as well as the effect on a non-target species. Entomopathogenic fungi were isolated from the Palamalai Hills, India, using the insect bait method, and the Metarhizium majus (MK418990.1) entomopathogen was identified using biotechnological techniques (genomic DNA isolation and 18S rDNA amplification). Bag-formulated fungal conidial efficacy (2.5 × 103, 2.5 × 104, 2.5 × 105, 2.5 × 106, and 2.5 × 107 conidia/ml) was evaluated against third instar larvae of Spodoptera frugiperda at 3, 6, 9, and 12 days of treatment, and acid and alkaline phosphatases, catalase, and superoxide dismutase enzymatic responses were evaluated at 3 days post-treatment. After 12 days of treatment, non-target assays on the earthworm Eudrilus eugeniae were performed using an artificial soil assay. Results of the bag formulated fungal conidial treatment showed that S. frugiperda had high susceptibility rates at higher concentrations (2.5 × 107 conidia/ml) of M. majus. Lower concentration of 2.5 × 103 conidia/ml caused 68.6% mortality, while 2.5 × 107 conidia/ml caused 100% mortality at 9 days post treatment. Investigation into enzymatic responses revealed that at 3 days post M. majus conidia exposure (2.5 × 103 conidia/ml), insect enzyme levels had significantly changed, with acid and alkaline phosphatases, and catalase enzymes significantly reduced and superoxide dismutase enzymes significantly raised relative to the control. After 12 days of treatment, no sublethal effects of M. majus conidia were observed on E. eugeniae, with no observed damage to gut tissues including lumen and epithelial cells, the nucleus, setae, coelom, mitochondria, and muscles. This study offers support for the use of fungal conidia in the target-specific control of insect pests.

Determination, distribution, and environmental fate of Bacillus thuringiensis spores in various honeybee matrices after field application as plant protection product.

The increasing use of Bacillus thuringiensis (Bt)-based plant protection products (PPPs) has recently raised some concerns regarding their environmental accumulation and possible chronic exposure of non-target species, including pollinators, to higher than expected doses. The exposure level of such microbial PPPs in bee's matrices under field conditions has not yet been described. Therefore, the current study aims at evaluating the realistic exposure level and comparing the distributions and persistence of Bt spores under field conditions. A field trial with spray application in oilseed rape (Brassica napus) as a representative bee-attractive crop was conducted. During the experimental period, different matrices, including honeybee-collected and -stored matrices as well as bee larvae and dead bees, were collected and analyzed using newly established methods. The concentration of Bt spores in the various matrices was quantified. The results show high levels of Bt spores in honey sac and pollen pellets with reduction over time but no reduction of Bt spores in the stored matrices within the colony, i.e., nectar and bee bread, over time. Our results show for the first time the exposure level of bees to Bt spores under realistic field conditions and are fundamentally important for assessing potential exposure and risks for pollinators.

Improving Contagion and Horizontal Transmission of Entomopathogenic Fungi by the White-Spotted Longicorn Beetle, Anoplophora malasiaca, with Help of Contact Sex Pheromone.

The white-spotted longicorn beetle, Anoplophora malasiaca, is one of the most destructive pests of horticultural crops and street trees. Effective controls are needed because the effect of marketed insecticides is limited. Entomopathogenic fungi offer a solution, and improving the rate of infection would be a breakthrough in this beetle's control. The combination of pathogenic fungi and the beetle's contact sex pheromone was suggested. The surface of the female body is covered with contact sex pheromone, which elicit male mating behavior. To develop a method for the practical control of this beetle, we evaluated the arrestant activity of female extract containing contact pheromone coated on a black glass model. Males presented with a coated model held on for 5 h (mean) during an 8-h experiment. In contrast, males presented with a control model held on for <0.3 h. Males that held onto coated models attached to fabric impregnated with conidia of the fungus Beauveria brongniartii picked up much conidia, which they then passed on to females during mating.

Activities of Select Enzymes Involved in Insecticide Resistance in Spinosad-Resistant and -Susceptible Strains of Musca domestica (Diptera: Muscidae).

A Musca domestica L. strain collected from Pakistan has recently been shown to be resistant to spinosad; however, there is scarce information about the mechanism of resistance. For this reason, we explored whether a metabolic-based mechanism was responsible by analyzing the activities of the metabolic detoxifying enzymes, carboxylesterases, glutathione S-transferases, and mixed-function oxidases, in both a spinosad-selected (Spin-SEL) strain of M. domestica and a susceptible counterpart (Lab-susceptible). The results revealed that both strains were statistically at par in terms of enzyme activities. The activity of carboxylesterases in the Lab-susceptible strain was 78.17 ± 3.06 in comparison to 79.16 ± 3.31 nmol min-1 mg-1 in the Spin-SEL strain. The activity of mixed-function oxidases was 51.58 ± 4.20 in the Lab-susceptible strain, whereas 54.33 ± 4.08 pmol min-1 mg-1 was recorded in the Spin-SEL strain. The activity of glutathione S-transferases was 86.50 ± 4.59 (Lab-susceptible) and 90.33 ± 2.81 nmol min-1 mg-1 (Spin-SEL). These results revealed that the studied enzymes might not be responsible for spinosad resistance in the studied strain of M. domestica. Therefore, studies should be extended to find out other possible mechanisms of spinosad resistance.

Molecular characterization of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Thailand.

A total of 511 local isolates of Bacillus thuringiensis from different geographical regions of Thailand were analyzed for the presence of the cry1A, cry1B, cry2A, cry9, and vip3A genes encoding for lepidopteran-specific toxins. PCR results revealed that 94.32% (482/511) of B. thuringiensis isolates harbored at least one of the detected genes, of which the cry1A, cry1B, cry2A, cry9, and vip3A genes were detected at frequencies of 90.61%, 89.63%, 76.32%, 40.70%, and 48.18%, respectively. Nineteen gene-combination profiles were discovered among 482 B. thuringiensis isolates, of which the most frequently detected profile contained the cry1A, cry1B, cry2A, and vip3A genes. Sixty-one isolates (12.66%), which harbored all of the detected insecticidal toxin genes, were further detected for the exochitinase (chi36) gene and chitinase activity. The results revealed that all 61 isolates contained the chi36 gene and exhibited chitinase activity. Insect bioassays showed that five isolates were highly toxic (more than 80% mortality) against second instar larvae of Spodoptera litura, of which the highest insect mortality (93%) was obtained from the B. thuringiensis isolates 225-15 and 417-1. Scanning electron microscopy revealed that the crystal morphologies of the five effective isolates were bipyramidal and cuboidal shapes. SDS-PAGE analysis of the spore-crystal mixture showed major bands of approximately 65 and 130 kDa. These five effective strains are alternative candidates for use as a microbial insecticide for the control of the S. litura pest.

Herbivore-Induced Defenses in Tomato Plants Enhance the Lethality of the Entomopathogenic Bacterium, Bacillus thuringiensis var. kurstaki.

Plants can influence the effectiveness of microbial insecticides through numerous mechanisms. One of these mechanisms is the oxidation of plant phenolics by plant enzymes, such as polyphenol oxidases (PPO) and peroxidases (POD). These reactions generate a variety of products and intermediates that play important roles in resistance against herbivores. Oxidation of the catecholic phenolic compound chlorogenic acid by PPO enhances the lethality of the insect-killing bacterial pathogen, Bacillus thuringiensis var. kurstaki (Bt) to the polyphagous caterpillar, Helicoverpa zea. Since herbivore feeding damage often triggers the induction of higher activities of oxidative enzymes in plant tissues, here we hypothesized that the induction of plant defenses would enhance the lethality of Bt on those plants. We found that the lethality of a commercial formulation of Bt (Dipel® PRO DF) on tomato plants was higher if it was applied to plants that were induced by H. zea feeding or induced by the phytohormone jasmonic acid. Higher proportions of H. zea larvae killed by Bt were strongly correlated with higher levels of PPO activity in the leaflet tissue. Higher POD activity was only weakly associated with higher levels of Bt-induced mortality. While plant-mediated variation in entomopathogen lethality is well known, our findings demonstrate that plants can induce defensive responses that work in concert with a microbial insecticide/entomopathogen to protect against insect herbivores.

Selection and characterization of Bacillus thuringiensis isolates with a high insecticidal activity against Spodoptera frugiperda (Lepidoptera: Noctuidae) / Seleção e caracterização de isolados de Bacillus thuringiensis com Alta atividade inseticida contra Spodoptera frugiperda (Lepidoptera: Noctuidae)

Spodoptera frugiperda (SMITH, 1797) (Lepidoptera: Noctuidae) affects diverse crops of great economic interest, for instance, it can cause severe yield losses in maize, rice and sorghum. In this study, a selection and characterization of Bacillus thuringiensis (BERLINER, 1911) isolates with a high insecticidal activity against S. frugiperda was performed. Fifty-two crystal-forming B. thuringiensis isolates that were identified from 3384 Bacillus-like colonies were examined and screened by PCR for the presence cry genes (cry1, cry1Aa, cry1Ab, cry1Ac, cry1D, cry2 and cry2Ab). Four isolates that showed high toxicity towards S. frugiperda were shown to harbor cry2 genes. The crystals were analyzed by electron microscopy and showed bipyramidal and cuboidal shapes. Furthermore, these four isolates had lethal concentration (LC50) values of 44.5 ng/cm2 (SUFT01), 74.0 ng/cm2 (SUFT02), 89.0 ng/cm2 (SUFT03) and 108 ng/cm2 (SUFT 04) to neonate S. frugiperda larvae. An ultrastructural analysis of midgut cells from S. frugiperda incubated with the SUFT01 spore-crystal complex showed disruptions in cellular integrity and in the microvilli of the midgut columnar cells. The isolates characterized in this work are good candidates for the control of S. frugiperda, and could be used for the formulation of new bioinsecticides.

Spodoptera frugiperda (SMITH, 1797) (Lepidoptera: Noctuidae) afeta diversas culturas de grande interesse econômico, por exemplo, pode causar severas perdas em milho, arroz e sorgo. Neste estudo, foi realizada uma seleção e caracterização de isolados de Bacillus thuringiensis (BERLINER, 1911) com elevada atividade inseticida contra S. frugiperda. Cinquenta e dois isolados formadores de cristal B. thuringiensis que foram identificados a partir de 3384 colônias foram examinados e testados por PCR para a presença dos genes cry (cry1, cry1Aa, cry1Ab, cry1Ac, cry1D, cry2 e cry2Ab). Quatro isolados que apresentaram alta toxicidade contra S. frugiperda foram mostrados para abrigar os genes cry2. Os cristais foram analisados por microscopia eletrônica e mostraram formas bipiramidais e cúbicas. Os valores da concentração letal (CL50) destes quatro isolados foram de 44,5 ng / cm2 (SUFT01), 74,0 ng / cm2 (SUFT02), 89,0 ng / cm2 (SUFT03) e 108 ng / cm2 (suft 04) para larvas recém-eclodidas de S. frugiperda. Uma análise ultra-estrutural das células do intestino médio de S. frugiperda incubadas com complexo esporo-cristal do isolado SUFT01 mostrou rupturas na integridade celular e microvilosidades das células cilíndricas do intestino médio. Neste estudo, o alto nível de atividade inseticida de isolados os torna excelentes candidatos para o controlo de S. frugiperda, e pode proporcionar alternativas no controle destas populações de pragas, bem como a formação de novos bioinsecticidas.

Distribution of Toxin Genes and Enterotoxins in Bacillus thuringiensis Isolated from Microbial Insecticide Products.

Bacillus thuringiensis microbial insecticide products have been applied worldwide. Although a few cases of B. thuringiensis foodborne illness have been reported, little is known about the toxigenic properties of B. thuringiensis isolates. The aims of this study were to estimate the pathogenic potential of B. thuringiensis selected from microbial insecticide products, based on its possession of toxin genes and production of enterotoxins. Fifty-two B. thuringiensis strains selected from four kinds of microbial insecticide products were analyzed. PCR assay for detection of toxin genes and immunoassay for detection of enterotoxins were performed. The hemolysin BL complex as a major enterotoxin was produced by 17 (32.7%), whereas the nonhemolytic enterotoxin complex was detected in 1 (1.9%) of 52 B. thuringiensis strains. However, cytK, entFM, and ces genes were not detected in any of the tested B. thuringiensis strains. The potential risk of food poisoning by B. thuringiensis along with concerns over B. thuringiensis microbial insecticide products has gained attention recently. Thus, microbial insecticide products based on B. thuringiensis should be carefully controlled.

Use of by-products rich in carbon and nitrogen as a nutrient source to produce Bacillus thuringiensis (Berliner)-based biopesticide / Produção de biopesticida à base de Bacillus thuringiensis (Berliner) usando meios alternativos ricos em carbono e nitrogênio como fontes de nutrientes

The amount and sources of carbon and nitrogen used to produce Bacillus thuringiensis (Berliner)-based biopesticide may infl uence the quality of the fi nal product. The objective of this research was to test different levels of carbon and nitrogen: medium 1 - 1.5% maize glucose + 0.5% soy fl our,medium 2 - 3.0% maize glucose + 1.0% soy fl our, medium 3 - 1.0% maize glucose + 3.0% soy fl our and medium 4 - Luria Bertani (LB) + salts (FeSO4, ZnSO4, MnSO4, MgSO4). The seed culture was produced in LB medium plus salt, under agitation (200 rpm) for 18h at 30oC. The strain 344 of Bt wasused (B. thuringiensis var tolworthi – belonging to the Embrapa´s Bt Bank). The pH was measured at regular intervals. and After culturing for 96h, the pH of the four tested media was basifi ed (6.91 and 8.15), the number of spores yielded 4.39 x 109 spores/ml in medium 3, where the amount of proteinis high. The dry biomass weight accumulated in media 3 was 39.3 g/l. Mortality of 2-day-old larvae Spodoptera frugiperda (J.E. Smith) was 100% when using Bt produced in media 3 and 4. CL50 for medium 3 was 8.4 x 106 spores/ml. All tested media were satisfactory to Bt growth, and medium 3wass the most promising to be used on a large scale Bt-based biopesticide production.

A quantidade de carbono e nitrogênio usados para produzir biopesticidas à base de Bacillus thuringiensis (Berliner) pode infl uenciar a qualidade fi nal do produto. O objetivo deste trabalho foi testar meios com diferentes níveis de carbono e nitrogênio: meio 1 - glicose de milho a 1.5% + farinha de soja a 0,5%, meio 2 - glicose de milho a 3,0% + farinha de soja a 1,0%, meio 3 - glicose de milho a 1,0% + farinha de soja a 3,0% e meio 4 - Luria Bertani (LB) + sais (FeSO4, ZnSO4, MnSO4 e MgSO4). O inóculo semente foi produzido usando 150 ml de meio LB mais sais incubados por 18h a 30oC, sob agitação de 200 rpm. A cepa utilizada foi 344 (B. thuringiensis var tolworthi – pertencente ao Banco de Microorganismos da Embrapa). O pH foi medido a intervalos regulares. Após 96h de cultivo, o pH de todos os meios testados tenderam ao básico (entre 6,91 e 8,15), o maior número de esporos foi de 4,39 x 109 sporos/ml no meio 3, onde o teor de proteína usado foi o mais alto. A produção de massa celular foi maior no meio 3, com total acumulado de 39,3 g/l. A mortalidade de larvas de Spodoptera frugiperda (J.E. Smith) de dois dias de idade nos meios 3 e 4 foi de 100%. A CL50 para o meio 3 foi de 8,4 x 106 esporos/ml. Os meios alternativos usados promoveram crescimento satisfatório de Bt, sendo o meio 3 o mais promissor para ser usadona produção de biopesticida à base de Bt.