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AISLAMIENTO Y CARACTERIZACIÓN MOLECULAR Y BIOLÓGICA DECEPAS NATIVAS DE Bacillus thuringiensis PARA EL CONTROL DETuta absoluta (Meyrick: Lepidoptera: Gelechiidae), INSECTO PLAGA DEL TOMATE(Lycopersicon esculentum)
Abstract
RESUMEN Se recolectaron 28 muestras de suelo en 14 municipios en Colombia. Se aislaron bacilos esporulados, que se caracterizaron microscópicamente cuantificando cristales. Los aislamientos positivos para cristales se sometie-ron a una caracterización bioquímica por medio de electroforesis de proteínas totales (SDS-PAGE). Las cepas positivas para la presencia de genes cry1 fueron sometidas a dos rondas de M-PCR con 2 mezclas de oligonu-cleótidos, reconociendo 6 genes específicos. Se aislaron 99 bacilos esporulados nativos que presentaron cristales con formas amorfas, bipiramidal, cuadradas, redondas y triangulares. Se observaron bacilos con 1, 2, 3 y 4 for-mas de cristal, estableciéndose 18 perfiles diferentes. Por SDS, se evidenciaron bandas de proteínas de 28 hasta 150 kDA clasificándose en 7 por su posible actividad biológica, lo que originó 28 perfiles diferentes. 35 bacilos esporulados presentaron genes cry1, y en estos, se detectaron por M-PCR los genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C y cry1D en el 76, 26, 21, 35, 32, y 8,8% respectivamente. De acuerdo a estas caracterizaciones se seleccionaron 10 aislamientos nativos como promisorios para el control de Tuta absoluta y se retaron contra larvas de 2do instar de este insecto plaga. Los bacilos nativos ZBUJTL39 y ZCUJTL11 presentaron una mejor actividad biológica que la cepa de referencia Bt var kurstaki HD1. El bacilo nativo ZCUJTL11 presentó una CL 50 de 2,4 µg/ml (P<0,05). La metodología estandarizada selecciona las cepas de acuerdo a su actividad bio-lógica potencial, como un paso previo a los ensayos biológicos, Este resultado es promisorio para posteriores investigaciones en ingeniería genética de tomate, para la obtención de cultivares autorresistentes a Tuta absoluta. ABSTRACT Were collected 28 soil samples in 14 municipalities in Colombia. Sporulated bacillus were isolated, characteri-zed microscopically by quantifying crystals. Isolates positive for crystals were subjected to biochemical charac-terization by electrophoresis of total proteins (SDS-PAGE). The strains positive for the presence of cry1 genes were subjected to two rounds of M-PCR with two oligonucleotide mixtures, recognizing specific genes 6. 99 bacillus were isolated forms filed with amorphous crystals, bipiramidal, square, round and triangular. Bacillus were observed with 1, 2, 3 and 4 forms of glass, with 18 different profiles. For SDS, showed protein bands of 28 to 150 kDa in 7 classified by their potential biological activity, resulting in 28 different profiles. 35 sporulated ba-cillus showed cry1 genes, and these were detected by M-PCR gene cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D in 76, 26, 21, 35, 32, and 8.8% respectively. According to these characterizations 10 isolates were selected as promising for the control of Tuta absoluta and challenged against the 2nd instar larvae of this insect pest. Strain ZBUJTL39 and introduced ZCUJTL11 better biological activity that the reference strain Bt. var kurstaki HD1. strain ZCUJTL11 presented a LC 50 of 2.4 mg / ml (P <0.05). The methodology selected strains according to their potential biological activity, as a preliminary step to biological tests, this result is promising for further research into genetic engineering of tomato cultivars to obtain resistant to Tuta absoluta.
... La estructura paraesporal cristalina se observó en 37 aislados de los 600, que indican la presencia de Bt, y la forma bipiramidal de esta inclusión paraesporal (Gitahy et al., 2007;Portela-Dussán et al., 2013;Salama et al., 2015) sólo se observó en 15 de los 37 aislados (Tabla 2), lo cual indicó que los aislados evaluados pertenecen al patotipo I de B. thuringiensis que son tóxicos para invertebrados, especialmente para larvas de los insectos de la orden Lepidoptera (Schnepf et al., 1998;Carreras, 2008;Sauka et al., 2010) 2008), que observaron mayor cantidad (89%) de cristales de forma bipiramidal en comparación que cualquier otra morfología. También concuerdan con otros autores (Höfte y Whitley, 1989;Baró et al., 2009;Ramírez et al., 2010;Azizoglu et al., 2011;Vázquez-Ramírez et al., 2015), quienes al analizar cepas nativas observaron una morfología de cristales bipiramidales típicos de la mayoría de las cepas pertenecientes a B. thuringiensis (Rabinovich et al. 2017). ...
CEPAS NATIVAS DE Bacillus thuringiensis CONTRA Spodoptera frugiperda y Alabama argillacea EN EL CULTIVO DE ALGODÓN (Gossypium barbadens) EN PIURA, PERÚ NATIVE STRAINS OF Bacillus thuringiensis AGAINST Spodoptera frugiperda and Alabama argillacea IN COTTON CROP (Gossypium barbadens) IN PIURA, PERU RESUMEN Los lepidópteros son una de las plagas más importantes del algodón (Gossypium barbadens). Su control se realiza con insecticidas sintéticos, que contaminan el medio ambiente, por lo que el control biológico constituye una alternativa ecológica al uso de tales productos químicos. El objetivo de esta investigación fue aislar cepas nativas de Bacillus thuringiensis (Bt) y evaluar su toxicidad contra Spodoptera frugiperda y Alabama argillacea, insectos plaga del cultivo de algodón. Las cepas nativas de Bt fueron aisladas de muestras de suelo de diferentes zonas algodoneras de Piura, Perú. Se obtuvieron 600 colonias del género Bacillus, de los cuales 37 cepas nativas presentaron características morfológicas similares a las cepas estándares de Bt (HD1 y NA118). La caracterización morfológica y bioquímica de las 37 cepas nativas mostró que 15 cepas presentaron cristal paraesporal de forma bipiramidal de acción tóxica contra lepidópteros, y características bioquímicas similares a las cepas estándares de Bt. El efecto tóxico de las 15 cepas nativas se evaluó con la prueba del alimento contaminado con discos de hojas de lechuga y algodón. Los resultados mostraron que las cepas IN-24, IN-30 e IN-34 ocasionaron 100% de mortalidad en S. frugiperda y las cepas IN-19, IN-24 e IN-25 ocasionaron 100% de mortalidad en A. argillacea, mientras que la cepa IN-24 ocasionó 100% de mortalidad en ambas especies. Por otra parte, la prueba de la dieta artificial reveló que las cepas IN-34, IN-01 e IN-31 ocasionaron los mayores porcentajes de mortalidad en S. frugiperda y A. argillacea, respectivamente. Se concluyó que existe varias cepas nativas de B. thuringiensis que pueden controlar S. frugiperda y A. argillacea. ABSTRACT The order Lepidoptera is one of the most important pests of cotton (Gossypium barbadens). These insects are controlled with synthetic insecticides, which contaminate the environment. Therefore, biological control is an environmentally friendly alternative to the use of such chemicals. The objective of this research was to isolate native strains of Bacillus thuringiensis (Bt) and evaluate their toxicity against Spodoptera frugiperda and Alabama argillacea, which are insect pests of the
... La estructura paraesporal cristalina se observó en 37 aislados de los 600, que indican la presencia de Bt, y la forma bipiramidal de esta inclusión paraesporal (Gitahy et al., 2007;Portela-Dussán et al., 2013;Salama et al., 2015) sólo se observó en 15 de los 37 aislados (Tabla 2), lo cual indicó que los aislados evaluados pertenecen al patotipo I de B. thuringiensis que son tóxicos para invertebrados, especialmente para larvas de los insectos de la orden Lepidoptera (Schnepf et al., 1998;Carreras, 2008;Sauka et al., 2010) 2008), que observaron mayor cantidad (89%) de cristales de forma bipiramidal en comparación que cualquier otra morfología. También concuerdan con otros autores (Höfte y Whitley, 1989;Baró et al., 2009;Ramírez et al., 2010;Azizoglu et al., 2011;Vázquez-Ramírez et al., 2015), quienes al analizar cepas nativas observaron una morfología de cristales bipiramidales típicos de la mayoría de las cepas pertenecientes a B. thuringiensis (Rabinovich et al. 2017). ...
The order Lepidoptera is one of the most important pests of cotton (Gossypium barbadens). These insects are controlled with synthetic insecticides, which contaminate the environment. Therefore, biological control is an environmentally friendly alternative to the use of such chemicals. The objective of this research was to isolate native strains of Bacillus thuringiensis (Bt) and evaluate their toxicity against Spodoptera frugiperd a and Alabama argillacea, which are insect pests of the cotton crop. The native strains of Bt were isolated from soil samples from different cotton areas of the Piura, Perú. 600 colonies of the genus Bacillus were obtained, of which 37 native strains presented morphological characteristics similar to the standard strains of Bt (HD1 and NA118). The morphological and biochemical characterization showed that 15 strains showed parasporal crystal of bipyramidal form of toxic action against Lepidoptera, exhibiting biochemical characteristics equal to the standard strains of Bt. The toxic effect of these 15 native strains was evaluated through a food poisoned test using lettuce and cotton leaf discs. The results showed that strains IN-24, IN-30 and IN-34 caused 100% mortality in S. frugiperda; strains IN-19, IN-24 and IN-25 caused 100% mortality in A. argillacea; while strain IN-24 caused 100% mortality in both species. The artificial diet test showed that strains IN-34, IN-01 and IN-31 caused the highest mortality rates in S. frugiperda and A. argillacea, respectively, when compared with the standard strains HD1 and NA118. It is concluded that there are several native strains of B. thuringiensis that can control S. frugiperda and A. argillacea.
Bacillus thuringiensis (Bt) es una bacteria entomopatógena utilizada en el control de insectos lepidópteros, coleópteros y dípteros plaga. Por la alta variabilidad genética en las proteínas Cry insecticidas, la caracterización de cepas de Bt representa un potencial para descubrir proteínas y actividades biológicas novedosas. En este estudio se realizó la caracterización microscópica, bioquímica y molecular de 20 cepas nativas de Bacillus thuringiensis pertenecientes al banco de cepas de la Universidad Jorge Tadeo Lozano (ujtl) con el fin de predecir su actividad biológica y su posible uso en control biológico. Las cepas caracterizadas se aislaron previamente en los departamentos de Boyacá (4), Cundinamarca (2), Huila (1) y Santander (5) y del ecosistema de manglar de la Ciénaga Grande de Santa Marta (8). Por microscopía de contraste de fases se observaron cristales amorfos (81.8%), triangulares, cuadrados y bipiramidales (18.2%). Utilizando pcr se identificaron entre 2-5 genes cry1 en 11 cepas nativas. Ocho cepas contienen los genes cry1Aa, cry1Ba y cry1Ca, (72.7%), cinco el gen cry1Ab (45.4%), tres el gen cry1Ac (27,2%) y únicamente dos cepas presentaron el gen cry1Da (18.1%). Se obtuvo el perfil electroforético de proteínas totales para las 11 cepas, encontrando proteínas con pesos moleculares entre 25-140 kDa e identificando cepas con perfiles electroforéticos con dos bandas (45.4%), tres (27.2%), cuatro (18.1%) y seis bandas de proteínas (9.1%). La secuenciación nucleotídica del gen arnr 16S arrojó que cinco cepas nativas seleccionadas al azar presentaron un porcentaje de similaridad por encima del 94% con relación a las secuencias depositadas en la base de datos del ncbi. De estas cuatro cepas, tres tuvieron un porcentaje de similaridad del 99%, 98% y del 97% con el género Bacillus sp (zsujtl67, zmujtl63 y zcujtl3, respectivamente) y las otras dos cepas mostraron porcentajes de similaridad del 96% y 94% con Bacillus thuringiensis (zmujtl94 y zmujtl96). De acuerdo con el análisis realizado, se relacionaron los espectros de susceptibilidad de 10 especies de insectos de importancia para la agricultura colombiana: cinco lepidópteros, tres dípteros y dos coleópteros frente a las 11 cepas nativas evaluadas, encontrándose que una de estas cepas presenta una posible actividad biológica sobre estas 10 especies de insectos (zmujtl94). La metodología utilizada permite predecir la actividad biológica de las cepas antes de la realización de ensayos biológicos en los que se emplean mucho tiempo y representan altos costos.
A method allowing rapid and accurate identification of different subgroups within the insecticidal crystal CryI protein-producing family of Bacillus thuringiensis strains was established by using PCR technology. Thirteen highly homologous primers specific to regions within genes encoding seven different subgroups of B. thuringiensis CryI proteins were described. Differentiation among these strains was determined on the basis of the electrophoretic patterns of PCR products. B. thuringiensis strains, isolated from soil samples, were analyzed by PCR technology. Small amounts of bacterial lysates were assayed in two reaction mixtures containing six to eight primers. This method can be applied to rapidly detect the subgroups of CryI proteins that correspond with toxicity to various lepidopteran insects.
A large plasmid containing all delta-endotoxin genes was isolated from Bacillus thuringiensis subsp. israelensis; restricted by BamHI, EcoRI, HindIII, KpnI, PstI, SacI, and SalI; and cloned as appropriate libraries in Escherichia coli. The libraries were screened for inserts containing recognition sites for BamHI, SacI, and SalI. Each was labeled with 32P and hybridized to Southern blots of gels with fragments generated by cleaving the plasmid with several restriction endonucleases, to align at least two fragments of the relevant enzymes. All nine BamHI fragments and all eight SacI fragments were mapped in two overlapping linkage groups (with total sizes of about 76 and 56 kb, respectively). The homology observed between some fragments is apparently a consequence of the presence of transposons and repeated insertion sequences. Four delta-endotoxin genes (cryIVB-D and cytA) and two genes for regulatory polypeptides (of 19 and 20 kDa) were localized on a 21-kb stretch of the plasmid; without cytA, they are placed on a single BamHI fragment. This convergence enables subcloning of delta-endotoxin genes (excluding cryIVA, localized on the other linkage group) as an intact natural fragment.
An 80-kDa parasporal crystal protein was detected in protein extracts of sporangia of Bacillus popilliae isolated from a diseased larva of the common cockchafer (Melolontha melolontha L.). Amino acid analysis of tryptic peptides revealed significant homology to the Cry2Aa endotoxins of Bacillus thuringiensis. The gene cryBP1 (cry18Aa1), which codes for the parasporal crystal protein, was found in a putative cry operon on the bacterial chromosome, which contains at least one further (smaller) open reading frame, orf1. The 706-amino-acid-long CryBP1 (Cry18Aa1) protein has a predicted molecular mass of 79 kDa and shows about 40% sequence identity to the Cry2 polypeptides of B. thuringiensis. In the light of published observations which suggest that the parasporal crystal proteins of B. popilliae are slightly toxic to their grub hosts, we propose the following survival strategy of B. popilliae. As an obligate pathogen of grubs, B. popilliae germinates in the gut of a grub and the parasporal crystal proteins are released and activated. The activated protein does not cause colloid osmotic lysis but instead damages the gut wall somehow to allow the vegetative cells to enter the hemolymph more easily. By becoming a parasite, B. popilliae can continue to proliferate efficiently while the living grub provides a food supply. This process is in contrast to that of B. thuringiensis, which rapidly kills the insect and is then limited to growth on the larval carcass.
During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.
Mexico is located in a transition zone between the Nearctic and Neotropical biogeographical regions and contains a rich and unique biodiversity. A total of 496 Bacillus thuringiensis strains were isolated from 503 soil samples collected from the five macroregions of the country. The characterization of the strain collection provided useful information on the ecological patterns of distribution of B. thuringiensis and opportunities for the selection of strains to develop novel bioinsecticidal products. The analysis of the strains was based on multiplex PCR with novel general and specific primers that could detect the cry1, cry3, cry5, cry7, cry8, cry9, cry11, cry12, cry13, cry14, cry21, and cyt genes. The proteins belonging to the Cry1 and Cry9 groups are toxic for lepidopteran insects. The Cry3, Cry7, and Cry8 proteins are active against coleopteran insects. The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal. The Cry11, Cry21, and Cyt proteins are toxic for dipteran insects. Six pairs of general primers are used in this method. Strains for which unique PCR product profiles were obtained with the general primers were further characterized by additional PCRs with specific primers. Strains containing cry1 genes were the most abundant in our collection (49.5%). Thirty-three different cry1-type profiles were identified. B. thuringiensis strains harboring cry3 genes represented 21.5% of the strains, and 7.9% of the strains contained cry11 and cyt genes. cry7, cry8, and cry9 genes were found in 0.6, 2.4, and 2.6% of the strains, respectively. No strains carrying cry5, cry12, cry13, cry14, or cry21 genes were found. Finally, 14% of the strains did not give any PCR product and did not react with any polyclonal antisera. Our results indicate the presence of strains that may harbor potentially novel Cry proteins as well as strains with combinations of less frequently observed cry genes.
Bacillus thuringiensis crystal proteins, well known to be toxic to certain insects but not pathogenic to mammals, are used as insecticidal proteins
in agriculture and forest management. We here identified a crystal protein that is non-insecticidal and non-hemolytic but
has strong cytocidal activity against various human cells with a markedly divergent target specificity, e.g. highly cytotoxic to HepG2 and Jurkat and less cytotoxic to the normal hepatocyte (HC) and HeLa. In slices of liver and colon
cancer tissues, the toxin protein preferentially killed the cancer cells, leaving other cells unaffected. The cytocidal effect
of the protein is non-apoptotic with swelling and fragmentation of the susceptible cells, although the apoptotic process does
occur when the cell damage proceeded slowly. The amino acid sequence deduced from the nucleotide sequence of the cloned gene
of the protein has little sequence homology with the insecticidal crystal proteins of B. thuringiensis. These observations raise the presence of a new group of the B. thuringiensis toxin and the possibility of new applications for the protein in the medical field.
Using PCR with a set of specific oligonucleotide primers to detect cryI-type genes, we were able to screen the cry-type genes of 225 Bacillus thuringiensis soil isolates from Taiwan without much cost in time or labor. Some combinations of cry genes (the cry-type profile) in a single isolate were unique. We identified five distinct profiles of crystal genes from the B. thuringiensis soil isolates from Taiwan. The cry genes included cryIA(a), cryIA(b), cryIA(c), cryIC, cryID, and cryIV. Interestingly, 501 B. thuringiensis isolates (93.5% of the total number that we identified) were isolated from areas at high altitudes. The profiles of cry-type genes were distinct in all isolation areas. The distribution of cry-type genes of our isolates therefore depended on geography. Using PCR footprinting to detect cryIC-type genes, we identified two distinct cryIC footprints from some of our isolates, indicating that these isolates may contain novel cryIC-type genes. B. thuringiensis isolates containing cryIA(a)-, cryIA(b)-, and cryIA(c)-type genes exhibited much greater activity against Plutella xylostella than did other isolates, indicating that multiple cry-type genes may be used as markers for the prediction of insecticidal activities.
A large number of Bacillus thuringiensis (Bt) isolates have been obtained from soil samples in China. The flagellar antigen serotypes, cry genes and crystal proteins of 570 Bt isolates were determined, and the pesticidal activity was assayed against the insects, Plutella xylostella, Heliothis armigera, Phaedon brassicae and Locusta migratoria manilensis, and the snail, Oncomelania hupensis. The results indicated that the Bt isolates were distributed within 35 H-serotypes, in which isolates of H3 were the most abundant (20%) followed by H5 (13%), H7 (9%) and H4 (8.7%), whereas isolates of other H-serotypes were less than 6%. The percentage of isolates containing the genes cry1Ac, cry1Aa/cry1Ac, cry1Aa/cry1Ac/cry1Ab, and cry1Aa/cry1Ac/cry1C was 14.7%, 6.6%, 5.6%, and 7.0%, respectively, while 265 isolates, representing 46.5% of the 570 Bt isolates, did not show any amplification product for the genes cry1Aa, cry1Ac, cry1C, cry2, cry3, cry4, cry7Aa. Some of the 570 Bt isolates caused high mortality of the assayed pests with 14.9%, 6%, 1.6%, 1.1%, and 0.2% of the isolates killing more than 90% of P. xylostella, H. armigera, P. brassicae, O. hupensis, and L. migratoria manilensis, respectively. The remaining 76.2% of the 570 isolates caused no mortality or less than 90% mortality against the tested insect and snail species.
A total of 6909 isolates of Bacillus thuringiensis were obtained from 455 samples of soil, insect larvae, and insect habitats from throughout New Zealand. Isolates tested for toxicity killed insects in three orders: lepidoptera, diptera and coleoptera. Some isolates were toxic to both Lepidoptera and Diptera and some were not toxic to any of the insects tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the different groups of toxic crystals indicated a diversity of crystal types.
Bacillus thuringiensiswas isolated from natural samples collected from 80 countries. The majority, 45% of the 5303 isolates, originated from stored products, with 25% originating from soil. The materials richest in isolates active in insects were mushroom compost and stored products. The proportion with bipyramidal-shaped crystals was 46%, while among the range of other shapes 14% were spherical and 4% rectangular. The largest proportion of isolates, 45%, was found in the United Kingdom with the majority originating from stored mills, which import produce from all over the world; 22% came from North America. Using single-dose assays, 44% of the isolates killed less than 25% of larvae of all four Lepidoptera tested,Heliothis virescens, Pieris brassicae, Spodoptera littoralis,andAgrotis ypsilon.Among isolates killing more than 25% of the larvae, every combination of activities of the 16 possible against these 4 insects was found, in proportions ranging from 0.6 to 14.5%, suggesting considerable diversity of insect activity. The 44% of strains with little or no activity to Lepidoptera (
24 strains ofBacillus thuringiensis type are studied by the determination of 35 cultural and biochemical characters. About 10 different biochmeical characters
make it possible to classify these strains into six major biochemical groups represented by one or two type-strains (biotypes).
On the other hand, the study of these 24 strains by the flagellate agglutination reaction also leads to six serological groups,
each one provided with a different H antigen, and which fit perfectly the biochemical classification. This fact emphasizes
the validity of this latter classification and, for the first time, brings in a double biochemicals-serological basis for
the classification of theB. thuringiensis type strains.
A classification for crystal protein genes of Bacillus thuringiensis is presented. Criteria used are the insecticidal spectra and the amino acid sequences of the encoded proteins. Fourteen genes are distinguished, encoding proteins active against either Lepidoptera (cryI), Lepidoptera and Diptera (cryII), Coleoptera (cryIII), or Diptera (cryIV). One gene, cytA, encodes a general cytolytic protein and shows no structural similarities with the other genes. Toxicity studies with single purified proteins demonstrated that every described crystal protein is characterized by a highly specific, and sometimes very restricted, insect host spectrum. Comparison of the deduced amino acid sequences reveals sequence elements which are conserved for Cry proteins. The expression of crystal protein genes is affected by a number of factors. Recently, two distinct sigma subunits regulating transcription during different stages of sporulation have been identified, as well as a protein regulating the expression of a crystal protein at a posttranslational level. Studies on the biochemical mechanisms of toxicity suggest that B. thuringiensis crystal proteins induce the formation of pores in membranes of susceptible cells. In vitro binding studies with radiolabeled toxins demonstrated a strong correlation between the specificity of B. thuringiensis toxins and the interaction with specific binding sites on the insect midgut epithelium. The expression of B. thuringiensis crystal proteins in plant-associated microorganisms and in transgenic plants has been reported. These approaches are potentially powerful strategies for the protection of agriculturally important crops against insect damage.
The biogenesis of the crystalline inclusion of Bacillus thuringiensis has been studied in the more general context of sporulation and in regard to the formation of some spore constituents. It has been shown that crystal protein is essentially formed during stages III and IV, before the acquisition of octanol resistance. One fraction of protein spore coats which is solubilized by a reducing agent associated with urea or guanidine hydrochloride, is synthesised at the same time; a more insoluble cystine-rich fraction is formed later, during stage V, just before the heat resistance is reached.
The analysis of bacterial extracts at intervals during sporogenesis indicates preferential synthesis of insoluble proteins (or proteins bound to membraneous structures), crystal protein represents a large proportion of these proteins. Immunological characterisation of crystal protein, with autoradiographic experiments reveals at least three antigens produced simultaneously after t4 and continuously throughout the biogenesis.
The sporulation of a mutant non crystal bearing strain was studied; it has been shown that this mutant accumulates soluble proteins having some of the properties of crystal constituents; in this case the synthesis and maturation of spore coats arise earlier in the course of sporulation than in the wild strain.
All results suggest a relation between crystal protein and a protein fraction of the spore coats.
To isolate a naturally occurring novel Bacillus thuringiensis strain, we investigated the distribution, toxicity, morphology, H serotype, and gene type of B. thuringiensis from residue samples of granary in Korea. A total of 163 B. thuringiensis isolates out of 411 samples producing spore and crystal were obtained. In toxicity tests, 80% of all isolates were toxic to lepidoptera, and 12% were not toxic to any of tested insects. And dipteran-active and lepidopteran/dipteran-active isolates were rare (2% and 6%, respectively). 152 B. thuringiensis isolates produced typical rhomboidal crystals, and the remainder produced parasporal inclusions with various morphologies. Serological test showed that B. thuringiensis isolates in granary represented 12 H serotypes, indicating varied distribution of B. thuringiensis. Of these, the serotype 3ab predominated, followed by the serotype 7 and 4ac. B. thuringiensis isolates of the serotype 3ab, 4ac, 5ab, 7, 8ab, 9, and 23 were toxic to lepidoptera, and the serotype 8bd, 12, 18, and 20ac were nontoxic, while 14 isolates were untypable by 33 B. thuringiensis H antisera. The frequency of toxicity against lepidoptera and diptera was primarily highly toxic. PCR analysis using cryI gene type-specific primers showed that cryIA(b) genes are frequently found and cryIE gene exists in only one isolate. Analysis of B. thuringiensis crystals and plasmid DNAs indicated a diversity of crystal and gene types.
The classification of Bacillus thuringiensis strains has been revised and updated based on flagellar antigens which have been in use for many years. Sixty-nine serotypes and 13 sub-antigenic groups have now been identified, giving 82 serovars among the 3500 B. thuringiensis isolates of the IEBC Collection. The number of serovars has gradually increased with the total number of strains. The biochemical characters used have also been investigated and their value assessed for identification of B. thuringiensis at the subspecies level. A crystal analysis was carried out in terms of morphology, delta-endotoxin profiles and larvicidal activity for the newly identified serovars. It was found that atypical crystals, some with novel components, are becoming more common. No insect susceptible to these serovars has been discovered among known target species. The number of cross-reacting H-antigens among B. cereus strains is increasing and may be of biological significance.
A Bacillus thuringiensis isolate, 89-T-34-22, belonging to the serovar shandongiensis (H22) produced noninsecticidal and nonhemolytic proteins crystallizing into irregular-shaped parasporal inclusions. The proteins showed in vitro cytotoxicity to human cells, including cancer cells, only when activated by protease treatment. The human leukemic T (MOLT-4) cells were > 100 times more susceptible than HeLa and normal T cells to the proteins of 89-T-34-22. The cytotoxicity was dose dependent and the median effective concentration for the MOLT-4 was 3.5 microg/ml. The cytopathy induced by the 89-T-34-22 proteins was characterized by remarkable condensation of the nucleus and cell-ballooning. Five major parasporal proteins of 89-T-34-22, with molecular masses in the range of 16-160 kDa, shared no similarity with the previously reported proteins in terms of the N-terminal sequence.
To identify and characterize Bacillus thuringiensis strains highly toxic to Spodoptera frugiperda, and to explore the genetic diversity of such strains.
The insecticidal activity of 1100 strains of B. thuringiensis from Colombian soil samples was assayed against first instar S. frugiperda larvae, and 32 active strains were found. After a second bioassay evaluation, the eight most potent strains were selected for further characterization, which included crystal protein profiles determined by polyacrylamide gel electrophoresis, plasmid profile, plasmid restriction patterns, cry gene composition, qualitative determination of beta-exotoxin production, random amplified polymorphic DNA, serotyping, and toxicity to S. frugiperda. All Colombian strains contained cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D genes. However, PCR profiles of the Colombian strains suggested the presence of variants of the cry1 genes. Serotyping indicated that these strains belong to the kurstaki, thuringiensis, canadiensis and indiana subspecies. Interestingly, three strains belonging to different serotypes and subspecies were found in the same soil sample, and toxicity ranged between 11 and 976 ng cm(-2) of diet.
It has been shown that B. thuringiensis strains belonging to different serotypes and displaying variable potency to S. frugiperda larvae can be found in the same soil sample.
The results obtained indicate that some of the B. thuringiensis strains studied could be of interest for further development for S. frugiperda control programmes.
We found the insect control agent Bacillus thuringiensis to be a ubiquitous soil microorganism. Using acetate selection to screen soil samples, we isolated B. thuringiensis in 785 of 1,115 soil samples. These samples were obtained in the United States and 29 other countries. A total of 48% of the B. thuringiensis isolates (8,916 isolates) fit the biochemical description of known varieties, while 52% represented undescribed B. thuringiensis types. Over 60% (1,052 isolates) of the isolates tested for toxicity were toxic to insects in the orders Lepidoptera or Diptera. Soil samples were collected from various habitats, including those habitats with different numbers of insects. The current presence of insects did not predict the presence of B. thuringiensis in a particular soil sample. B. thuringiensis was most abundant in samples from Asia.