Diouneia Lisiane Berlitz

Toxicidade via intragástrica e intraperitoneal de Bacillus thuringiensis e Melia azedarach, em camundongos

The aim of this investigation was the assessment of toxicity of two new isolates of Bacillus thuringiensis, and the aqueous extract of Melia azedarach through in vivo assays in CF1 mice. Bt 1958-2, Bt 2014-2 and the BTh Thuricide 63 standard isolates were grown in liquid usual glicosed medium, and Cry proteins were purified by centrifugation on a sucrose gradient. The supernatant was autoclaved at 121o C, 15min. to maintain the exotoxins. Dehydrated leaves of M. azedarach were used to prepare a 10% aqueous extract. Mice were treated either orally or intraperitoneally with a whole bacterial suspension (1.1010 UFC/mL), a culture supernatant or purified crystal protein (50 µg/mL), and with the plant extract (50 µg/mL). The stomachs of the mice were collected and observed in stereomicroscopy, and the stomach contents were analyzed in 10% SDS-PAGE. Results showed that none of the oral treatments were toxic to mice, but intraperitoneal bacterial suspensions were lethal to the animals 6 - 24 hours after injection. In conclusion, the Cry proteins of the new B. thuringiensis isolates must be evaluated for their use as tools in the biotechnology field, since they do not show toxicity against mammals, intragastrically or peritoneally, just like the M. azedarach aqueous extract (10%), with those being indicated for the biological control of pest insects.

Toxicology of the Bioinsecticides Used in Agricultural Food Production

As populations grow in numbers, the demands for food production increase and are generally met by the intensification of livestock breeding and the increase of agricultural activities. This in turn increases the quantity of chemical pesticides required to control the losses in production caused by insect pests preying on the food plants and disturbing the animals. Once applied these pesticides may cause resistance to the synthetic molecules, contaminate biotic and abiotic components like plants, soil, water and/or the local water network and can also effect non-targeted organisms, such as fish, small mammals, birds and so on.

Frequência de Bacillus spp. em solos de diferentes sistemas de cultivo de arroz irrigado em Cachoeirinha, RS

Entomopathogenic Bacillus are bacteria found mainly in the soil and are considered important agents in biological control of agricultural diseases and pests. However, there are fen studies describing the influence that different agricultural practices have on Bacillus spp. populations. This study proposes to determine the occurrence frequency of entomopathogenic bacteria from Bacillus genus in soil samples. These were collected during the agricultural year 2006/07 from different irrigated rice cultivation systems used in the EEA-IRGA plantations in Cachoerinha, State of Rio Grande do Sul, Brazil. We analyzed 36 soil samples taken from three irrigated rice plantation systems; (i) Pre-germinated (SPG); (ii) Conventional (SPC); and (iii) Direct plantation (SPD) and a fourth sample from a non-planted area (C). In each test one gram of soil was diluted, pasteurized and inoculated in Nutrient Agar (NA) and incubated for 24 hours at 30°C. All the bacteria colony-forming units (CFU) were transplanted to selective mediums and the B. thuringiensis, B. sphaericus and B. cereus species were identified using a phase contrast microscope. No significant differences in the occurrence frequencies were found in the soil samples of all plantation systems analyzed. On the other hand, the Bacillus spp frequency varied in the sampled period during the different growing phases of the irrigated rice in the EEA-IRGA plantations evaluated. Irrigation was the main factor contributing to variation of the Bacillus in the plantations.

Toxicology effects of δ-endotoxins and β-exotoxins of Bacillus thuringiensis in Wistar rats

Bacillus thuringiensis is an entomopathogen consisting of toxic proteins, such as the δ- endotoxins specific to insects. However, some subspecies can produce β-exotoxins that are nonspecifi c and toxic to the vertebrates. So, in this research the toxicology effect of two isolates of B. thuringiensis in Wistar rats have been assessed. In the in vivo assays the animals have been given 3.1010UFC/mL of B. thuringiensis thuringiensis and B. thuringiensis aizawai and supernatant bacterial suspensions orally. The excrements and the stomachic content of the animals have been collected and analyzed in SDS-PAGE (10%). The analyzed data show protein fragments between 151 kDa and 28 kDa, seeing that the fecal samples show only one track of peptides in the treatments when compared with the control group. The assessments of the stomach of the treated rats carried out under stereomicroscope have not shown alterations when compared with the control. These results indicate that the toxins which can be found in both B. thuringiensis species used in these assays can be degraded by the gastrointestinal conditions of the animals and have not presented oral toxic effect to this mammalian species under the conditions which the experiments had been carried out. Key words: bacterium, toxins, mammalian, toxicity.

Bacillus thuringiensis Characterization: Morphology, Physiology, Biochemistry, Pathotype, Cellular, and Molecular Aspects

In this publication, “Bacillus thuringiensis and Lysinibacillus sphaericus – characterization and use in the field of biocontrol,” this chapter can be seen as a brief general and historical introduction to the central theme of the book, where data on the cellular physiology, biochemical, genetic, molecular, and toxicological aspects of the bacterium, B. thuringiensis (Bt), are reported. This predominant entomopathogenic prokaryote was discovered and denominated Bt around a century ago, between 1902 and 1911. From the microbiological point of view, this bacterium is ubiquitous, Gram-positive, produces ellipsoidal but predominantely cilindrical endospores (central to paracentral) and contains a parasporal inclusion body called crystal or δ-endotoxin. The crystal is constituted of Cry proteins with molecular weight between 30 kDa and 140 kDa, which are coded by cry genes. On the other hand, this bacterial species synthesizes several enzymes and toxins that give them a wide adaptation to natural habitats. Bt strains have been studied and, over time, characterized and described as toxic and specific for Lepidoptera, Diptera, Coleoptera, Nematoda, Protozoa, Trematoda, Acari, Hymenoptera, Hemiptera, Orthoptera, Isoptera, Mallophaga, and among other target pests. Globally, 82 Bt serovars sometimes called subspecies were described until 1999, which currently correspond to more than 700 cry genes distributed in about 70 classes. The nomenclature review of cry genes, which encode Bt Cry proteins, has been published by Crickmore et al. and has been constantly updated on the website: http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/.

Bacillus thuringiensis: Different Targets and Interactions

In the microbial control of pests, the entomopathogen Bacillus thuringiensis offers the best biological alternative to chemical insecticides, either alone or in combination with other methods of field control, and is also a source of genes for the genetic engineering of plants. In this work, aspects related to new targets of this bacterium are described such as: Acromyrmex spp.; Nasutitermes ehrhardt; Euschistus heros; Oryzophagus oryzae; Blatella germanica; Pyricularia grisea, Rhizoctonia solani, Fusarium oxysporum; Fusarium solani and Meloidogyne spp. Also discussed are the interactions of Bacillus thuringiensis and B. subtilis with other biological control agents: Purpureocillium lilacinus; Campoletis flavicincta; Nuclear Polyhedrosis Virus; plant extracts and essential oils from medicinal plants. Data from our research group of Microbiology and Toxicology in Agroecosystems (MToxAgro/CNPq), as well as collaborating researchers of some public and private institutions of Brazil will be presented.

Bacillus and Biopesticides in Control of Phytonematodes

Currently the major challenge of humanity is focused on population growth/increase through agricultural production in order to meet the demand for food. Overtime, different pests have emerged, with some being of great importance. Among these pests, the nematodes are noted for attacking leguminous plants, grasses, citrus, and other fruits. The main pest species are of the genus Heterodera, Meloidogyne, Pratylenchus, and Globodera. These nematodes cause losses up to 100 %, preventing agriculture of certain areas. Financially, about

Mortality of Oryzophagus oryzae (Costa Lima, 1936) (Coleoptera: Curculionidae) and Spodoptera frugiperda (J E Smith, 1797) (Lepidoptera: Noctuidae) Larvae Exposed to Bacillus thuringiensis and Extracts of Melia azedarach

100 billion annual damage is caused by nematodes. These amount to 90 % of the yield of cotton, yams, beans, and soybeans, and in citrus damage is estimated at 14 % of production. Alternative methods of control are being studied, and in this context, a bacterium of the genus Bacillus has prominence and importance. Besides Bacillus subtilis, some by pesticides are already marketed for the control of nematodes, such as Bioarc® the basis of Bacillus megaterium, Bio Zeid® the basis of Trichoderma album, and also using the brown alga, Ascophyllum nodosum (Algaefol®), among others. The objective of this chapter is to report the use of different Bacillus species and some biopesticides used to control nematodes of agricultural importance.