The chronicle of the ups and downs that made Bacillus thuringiensis a natural insecticide

Bacillus thuringiensis is a worldwide known bacterium for its capacity to control insect pests thanks to the action of its parasporal crystal. The objective of this paper deals with the history, in some cases unknown, of the study of Bacillus thuringiensis that led it to be a crucial biological alternative in controlling pest insects. How the mode of action for killing insects was understood, as well as the field tests that were carried out to evaluate its effectiveness and to develop the first commercial products, are reflected in this review that presents and discusses the scientific successes and failures that marked the course of B. thuringiensis.

Bacillus thuringiensis es una bacteria conocida mundialmente por su capacidad para controlar insectos plaga, gracias a la acción de su cristal parasporal. El objetivo de esta revisión trata de la historia, en algunos casos desconocida, del estudio de Bacillus thuringiensis que la llevó a ser una importante alternativa biológica en el control de insectos plaga. Cómo se llegó a comprender el modo de acción para matar insectos, así como las pruebas de campo que se realizaron para evaluar su efectividad y lograr desarrollar los primeros productos comerciales están plasmados en esta revisión que presenta y discute los aciertos y desaciertos científicos que marcaron el rumbo de B. thuringiensis.

Evaluación antimicrobiana de un extracto metanólico de Beauveria bassian a contra bacterias patógenas de importancia nosocomial / Performance Evaluation of Maize Starch as a Multifunctional Excipient in Metronidazole Tablet Formulation

Objetivo: Se evaluó la actividad antimicrobiana de un extracto crudo de B. bassiana y dos fracciones del mismo contra bacterias de importancia clínica. Métodos: El micelio de cepa B. bassiana se remojó en metanol durante una semana, después se evaporo en un rotovapor a 45°C aplicando vacío. El extracto metanólico se hizo pasar con dos fases móviles para obtener una fracción A y B. La fracciones A, B y el extracto crudo C se evaluaron contra las cepas Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella tiphy, Pseudomonas aeruginosa y Acinetobacter baumannii con la técnica de microdilución en placa. Resultados: En la fracción A se observó un efecto antimicrobiano contra Salmonella typhi, Pseudomonas aeruginosa y Acinetobacter baumannii el crecimiento bacteriano alcanzó el 70, 60 y 83 % respectivamente. La fracción B causó un efecto antimicrobiano en Klebsiella pneumoniae, S. typhi, P. aeruginosa y A. baumannii con un crecimiento bacteriano del 62, 58, 41 y 7 % respectivamente. Y El extracto crudo no causó inhibición del crecimiento en A. baumannii, pero para el resto de la bacterias hubo un crecimiento del 56 al 88 %. Conclusiones: Beauveria bassiana es un hongo entomopatógeno que produce diferentes metabolitos con actividad insecticida, citotóxica, antifúngica, antibiótica y antiviral. Este es el primer estudio de los efectos antimicrobianos de un extracto metanólico del hongo entomopatógeno B. bassiana contra cepas bacterianas de importancia clínica

Objective: The antimicrobial activity of a crude extract of B. bassiana and two fractions thereof against clinically important bacteria was evaluated. Methods: The mycelium of strain B. bassiana was soaked in methanol for a week, then it was evaporated in a rotovap at 45 ° C applying a vacuum. The methanolic extract was passed through two mobile phases to obtain a fraction A and B. Fractions A, B and crude extract C were evaluated against the strains Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella tiphy, Pseudomonas aeruginosa and Acinetobacter baumannii with the plate microdilution technique. Results: In fraction A an antimicrobial effect against Salmonella typhi, Pseudomonas aeruginosa and Acinetobacter baumannii was observed, bacterial growth reached 70, 60 and 83% respectively. Fraction B caused an antimicrobial effect in Klebsiella pneumoniae, S. typhi, P. aeruginosa and A. baumannii with a bacterial growth of 62, 58, 41 and 7% respectively. And the crude extract did not cause growth inhibition in A. baumannii, but for the rest of the bacteria there was a growth of 56 to 88%. Conclusions: Beauveria bassiana is an entomopathogenic fungus that produces different metabolites with insecticidal, cytotoxic, antifungal, antibiotic and antiviral activity. This is the first study of the antimicrobial effects of a methanolic extract of the entomopathogenic fungus B. bassiana against bacterial strains of clinical importance

Haplotype Profile Comparisons Between Spodoptera frugiperda (Lepidoptera: Noctuidae) Populations From Mexico With Those From Puerto Rico, South America, and the United States and Their Implications to Migratory Behavior.

Fall armyworm [Spodoptera frugiperda (J. E. Smith)] is a major economic pest throughout the Western Hemisphere of maize, cotton, sorghum, and a variety of agricultural grasses and vegetable crops. Previous studies demonstrated extensive annual migrations occurring as far north as Canada from overwintering locations in southern Florida and Texas. In contrast, migratory behavior in the rest of the hemisphere is largely uncharacterized. Understanding the migration patterns of fall armyworm will facilitate efforts to predict the spread of pesticide resistance traits that repeatedly arise in this species and assess the consequences of changing climatic trends on the infestation range. Four independent fall armyworm colonies derived from widely separated populations in Mexico and two field collections were examined for their mitochondrial cytochrome oxidase I (COI) gene haplotypes and compared with other locations. The Mexico populations were most similar in their haplotype profile to those from Texas and South America, but also displayed some distinctive features. The data extend the haplotype distribution map in the Western Hemisphere and confirm that the previously observed regional differences in haplotype frequencies are stable over time. The Mexico collections were associated with haplotypes rarely found elsewhere, suggesting limited migratory interactions with foreign populations, including those in neighboring Texas.

Ruta graveolens Extracts and Metabolites against Spodoptera frugiperda.

The biological activity of Ruta graveolens leaf tissue extracts obtained with different solvents (ethyl acetate, ethanol, and water) and metabolites (psoralen, 2- undecanone and rutin) against Spodoptera frugiperda was evaluated. Metabolites levels in extracts were quantified by HPLC and GC. Ethyl acetate and ethanol extracts showed 94% and 78% mortality, respectively. Additionally, psoralen metabolite showed a high mortality as cypermethrin. Metabolite quantification in extracts shows the presence of 2-undecanone (87.9 µmoles mg(-1) DW), psoralen (3.6 µmoles mg(-1) DW) and rutin (0.001 pmoles mg(-1) DW). We suggest that these concentrations of 2-undecanone and psoralen in R. graveolens leaf tissue extracts could be responsible for S. frugiperda mortality.

Correlation between Pr1 and Pr2 gene content and virulence in Metarhizium anisopliae strains.

Metarhizium anisopliae is a widely studied model to understand the virulence factors that participate in pathogenicity. Proteases such as subtilisin-like enzymes (Pr1) and trypsin-like enzymes (Pr2) are considered important factors for insect cuticle degradation. In four M. anisopliae strains (798, 6342, 6345, and 6347), the presence of pr1 and pr2 genes, as well as the enzymatic activity of these genes, was correlated with their virulence against two different insect pests. The 11 pr1 genes (A, B, C, D, E, F, G, H, I, J, and K) and pr2 gene were found in all strains. The activity of individual Pr1 and Pr2 proteases exhibited variation in time (24, 48, 72, and 96 h) and in the presence or absence of chitin as the inductor. The highest Pr1 enzymatic activity was shown by strain 798 at 48 h with chitin. The highest Pr2 enzymatic activity was exhibited by the 6342 and 6347 strains, both grown with chitin at 24 and 48 h, respectively. Highest mortality on S. exigua was caused by strain 6342 at 48 h, and strains 6342, 6345, and 6347 caused the highest mortality 7 days later. Mortality on Prosapia reached 30% without variation. The presence of subtilisin and trypsin genes and the activity of these proteases in M. anisopliae strains cannot be associated with the virulence against the two insect pests. Probably, subtilisin and trypsin enzyme production is not a vital factor for pathogenicity, but its contribution is important to the pathogenicity process.

Characterization of the chitinase gene in Bacillus thuringiensis Mexican isolates.

The chitinase gene was molecularly characterized in five Bacillus thuringiensis Mexican isolates, MR10, MR11, MR21, MR33, and RN52. The proteins derived from these genes were tested for their chitinase activity using fluorogenic chitin derivatives. In order to verify if chitinase genes were functional, they were cloned, and enzymatic activity of recombinant chitinases was also tested. Results indicated that enzymes exhibited endochitinase activity. The highest hydrolytic activity shown against the chitin tetrameric derivative occurred at pH value of 6.5, and the optimum activity temperature was around 60 °C. The recombinant endochitinases showed a molecular mass of ∼77 kDa with isoelectric points from 6.5 to 7.0. Analysis of the nucleotide sequences showed highly conserved sequences among all isolates (97-99 %). Gene sequence analysis revealed a putative promoter (-35 TTGAGA and -10 TTAATA) and a Shine-Dalgarno sequence (5´-AGGAGA-3´) upstream from the open reading frame. The deduced amino acid sequence revealed that the proteins are modular enzymes composed by a family 18 glycosyl hydrolase domain located between amino acids 134 and 549, a fibronectin-binding domain (580 through 656), and a chitin-binding domain (664 through 771). The deduced amino acid sequences of our isolates showed a similarity close to 100 % respect to the sequences reported in the GenBank database.

Genetic differentiation among Maconellicoccus hirsutus (Hemiptera: Pseudococcidae) populations living on different host plants.

The pink hibiscus mealybug Maconellicoccus hirsutus (Green) is a dangerous pest that damages a wide variety of agricultural, horticultural, and forestry crops. Amplified fragment length polymorphism (AFLP) fingerprints were used to characterize the genetic variation of 11 M. hirsutus populations infesting three plant species in Nayarit, Mexico. Analysis was carried out using four primers combinations, producing 590 polymorphic bands. Cluster analysis, as well as bootstrap dendrogram and nonmetric multidimensional scaling analysis, grouped M. hirsutus populations according to their host plant. The estimated F(ST) values indicated a high differentiation in M. hirsutus populations among the three host plant species. These results were also supported by a Bayesian analysis, which indicated a population clustering robustness according to their host plant. Genetic variation among populations is not caused by geographic distances, as shown by a Mantel test.

Design of a Bacillus thuringiensis-based formulation that increases feeding preference on Spodoptera exigua (Lepidoptera: Noctuidae) larvae.

A Bacillus thuringiensis (Bt)-based formulation intended to increase feeding response in Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) was developed. Eight matrices were prepared from natural ingredients and confronted with fresh maize leaves to select one highly palatable to larvae. The selected matrix was combined with Bt HD-125 strain at three different concentrations (30, 70, and 100 g/kg) of the spore-crystal complex to elaborate three insecticidal formulations. Effects caused by the matrix, including palatability, any adverse effect, and reduction in toxic activity of the strain, were tested on the formulations. Results indicated that the blend of modified cornstarch, porcine gelatin, and dried corncob was the most preferred by larvae. Formulations obtained were evaluated in mortality bioassays with neonate larvae in laboratory, using artificial diet, and in the greenhouse, with maize, Zea mays L., plants to compare their effectiveness when applied to natural food. In laboratory tests, all formulations, including a commercial formulation, were ingested by larvae causing high mortality in neonate larvae; in the greenhouse tests, formulations were also ingested causing mortality in larvae, overcoming commercial formulation effectiveness. Results suggest that the selected matrix combined with Bt 70 g/kg is the most suitable formulation to achieve effective control because this concentration acts faster than the lower concentration and is equal than the higher concentration. This formulation, designed specifically against a particular pest, is highly effective and offers a high potential for effective pest control.

Biopesticide production from Bacillus thuringiensis: an environmentally friendly alternative.

Since its discovery as a microbial insecticide, Bacillus thuringiensis has been widely used to control insect pests important in agriculture, forestry, and medicine. The wide variety of formulations based on spore-crystal complexes intended for ingestion by target insects, are the result of many years of research. The development of a great variety of matrices for support of the spore-crystal complex enables many improvements, such as an increase in toxic activity, higher palatability to insects, or longer shelf lives. These matrices use many chemical, vegetable or animal compounds to foster contact between crystals and insect midguts, without harming humans or the environment. Biotechnology companies are tasked with the production of these kinds of bioinsecticides. These companies must not only provide formulations tailored to specific crops and the insect pests, but they must also search for and produce bioinsecticides based on new strains of high potency, whether wild or genetically improved. It is expected that new products will appear on the market soon, providing an increased activity spectrum and applicability to many other pest-impacted crops. These products may help develop a more organic agriculture. This review article discusses recent patents related to bioinsecticides.

Laboratory and field tests of spray-dried and granular formulations of a Bacillus thuringiensis strain with insecticidal activity against the sugarcane borer.

Formulations of Bacillus thuringiensis Berliner (Bt) with insecticidal activity against the sugarcane borer, Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae), were developed and tested under laboratory and field conditions. The formulations were prepared using biodegradable polymers such as modified corn starch as an encapsulating agent, gelatin as an adherent, powdered sugarcane as a feeding stimulant and a Bt var. kurstaki GM-34 strain from a non-sugarcane region as the active ingredient. The spore-crystal complex of this strain was mixed at three different concentrations (30, 70 and 100 g kg(-1)) with the other ingredients. The blends were prepared as spray-dried and granular formulations, and then submitted to laboratory tests with two day old larvae of D. saccharalis and field tests in sugarcane crops with natural sugarcane borer infestation. Spray-dried formulations in laboratory bioassays caused mortality near 100% with all three concentrations, and granular formulations caused mortality around 84%. The field tests showed that spray-dried formulations at 70 and 100 g kg(-1) concentrations were as effective as a commercial bioinsectide (Lepinox), while granular formulations were ineffective.