Bacillus thuringiensis: a natural endophytic bacterium found in wild plants.

Despite the fact that Bacillus thuringiensis is the most widely used bacterium in biological pest control, its ecology has been notoriously neglected. Its role in nature is uncertain, and a defined habitat and niche are under discussion. In this report, wild-type strains were isolated from the inner plant tissues as natural endophytic bacteria in wild plants. Once a reliable superficial sterilization technique was standardized, leaf samples from 110 wildlife plant species within 52 families were processed to obtain their endophytic microflora, which were able to grow in artificial media. From 93 morphologically different isolates, 22 showed the typical sporangium morphology of B. thuringiensis (endospore and parasporal bodies). These isolates were identified and characterized by their 16S ribosomal RNA, hag gene, MLST, and cry gene sequences. Also, isolates were characterized by Bc-RepPCR and parasporal body protein content. All the isolates showed at least some of the typical B. thuringiensis features tested, but 10 showed information in all those features, which, in a rigorous selection, were taken as B. thuringiensis sensu stricto strains. Only three subspecies were identified: five kurstaki, four nigeriensis, and one thuringiensis. None showed toxicity against mosquito larvae or Caenorhabditis elegans, and only one showed significant toxicity against Manduca sexta larvae. The role of B. thuringiensis as a natural endophytic bacterium is discussed.

Structural changes upon membrane insertion of the insecticidal pore-forming toxins produced by Bacillus thuringiensis.

Different Bacillus thuringiensis (Bt) strains produce a broad variety of pore-forming toxins (PFTs) that show toxicity against insects and other invertebrates. Some of these insecticidal PFT proteins have been used successfully worldwide to control diverse insect crop pests. There are several studies focused on describing the mechanism of action of these toxins that have helped to improve their performance and to cope with the resistance evolved by different insects against some of these proteins. However, crucial information that is still missing is the structure of pores formed by some of these PFTs, such as the three-domain crystal (Cry) proteins, which are the most commercially used Bt toxins in the biological control of insect pests. In recent years, progress has been made on the identification of the structural changes that certain Bt insecticidal PFT proteins undergo upon membrane insertion. In this review, we describe the models that have been proposed for the membrane insertion of Cry toxins. We also review the recently published structures of the vegetative insecticidal proteins (Vips; e.g. Vip3) and the insecticidal toxin complex (Tc) in the membrane-inserted state. Although different Bt PFTs show different primary sequences, there are some similarities in the three-dimensional structures of Vips and Cry proteins. In addition, all PFTs described here must undergo major structural rearrangements to pass from a soluble form to a membrane-inserted state. It is proposed that, despite their structural differences, all PFTs undergo major structural rearrangements producing an extended α-helix, which plays a fundamental role in perforating their target membrane, resulting in the formation of the membrane pore required for their insecticidal activity.

Isolation and characterization of two highly insecticidal, endophytic strains of Bacillus thuringiensis.

Recent discovery of endophytic strains of Bacillus thuringiensis significantly improves the knowledge on its ecology. It also may be a new source for the isolation of insecticidal strains. This report shows the characterization of two endophytic, highly insecticidal strains of B. thuringiensis. Strains LBIT-1250L and LBIT-1251P were isolated from lavender and Poinsettia sap, respectively. Their parasporal crystals were very similar in morphology to those shown by serotypes israelensis and kurstaki, respectively. Bioassays on Aedes aegypti fourth instar larvae and on Manduca sexta first instar larvae, respectively, showed significantly higher levels of toxicity than those of their standard counterparts, IPS-82 (israelensis) and HD-1 (kurstaki) strains, respectively. Characterization of both strains included the sequencing of flagellin (hag) gene, plasmid and Bc Rep-PCR patterns and crystal protein content. All four characterization features indicated that LBIT1250L is highly related to the IPS-82 standard (serotype H-14: israelensis); while the LBIT-1251P was highly related to the HD-1 standard (serotype H-3a3b3c kurstaki). These results indicate that endophytic strains of B. thuringiensis may be a new source of potential insecticidal strains and opens more in-depth studies about the role of this bacterium in such a specialized habitat.

Selection and characterization of two Bacillus thuringiensis strains showing nematicidal activity against Caenorhabditis elegans and Meloidogyne incognita.

Bacillus thuringiensis has been widely used as a biological control agent against insect pests. Additionally, nematicidal strains have been under investigation. In this report, 310 native strains of B. thuringiensis against Caenorhabditis elegans were tested. Only the LBIT-596 and LBIT-107 strains showed significant mortality. LC50s of spore-crystal complexes were estimated at 37.18 and 31.89 µg/mL for LBIT-596 and LBIT-107 strains, respectively, while LC50s of partially purified crystals was estimated at 23.76 and 20.25 µg/mL for LBIT-596 and LBIT-107, respectively. The flagellin gene sequence and plasmid patterns indicated that LBIT-596 and LBIT-107 are not related to each other. Sequences from internal regions of a cry5B and a cyt1A genes were found in the LBIT-596 strain, while a cry21A, a cry14A and a cyt1A genes were found in the LBIT-107 strain. Genome sequence of the LBIT-107 strain showed new cry genes, along with other virulence factors, hence, total nematicidal activity of the LBIT-107 strain may be the result of a multifactorial effect. The highlight of this contribution is that translocation of spore-crystal suspensions of LBIT-107 into tomato plants inoculated at their rhizosphere decreased up to 90% the number of galls of Meloidogyne incognita, perhaps the most important nematode pest in the world.