Susceptibility of agricultural pests of regional importance in South America to a Bacillus thuringiensis Cry1Ia protein.

Bacillus thuringiensis toxins of the Cry1I class have dual specificity for insects in the orders Coleoptera and Lepidoptera. We assessed the toxicity of a Cry1Ia protein from an Argentinian B. thuringiensis strain against agricultural pests in the families Tenebrionidae, Curculionidae, Noctuidae and Tortricidae. Three recombinant protein variants were produced that differed in length and fusion tag position to rule out artifactual results. The protein was toxic to Cydia pomonella and Rachiplusia nu. In contrast, Alphitobius diaperinus, Anthonomus grandis and Spodoptera frugiperda were not susceptible. The results are discussed with respect to previous studies and the prospective use of Cry1Ia in strategies to control major cotton pests in the region.

Paenibacillus xylanivorans sp. nov., a xylan-degrading bacterium isolated from decaying forest soil.

A xylanolytic bacterial strain, named A59T, was isolated from a forest soil consortium in southern Argentina. Strain A59T is a Gram-stain-positive, facultative anaerobic, endospore-forming and rod-shaped bacterium. Its optimal growth conditions are 30 °C (range, 28-37 °C), pH 7 (range, pH 5-10) and it tolerates up to 7 % of NaCl (range, 2-7 %). Chemotaxonomic analysis revealed that strain A59Tpossesses meso-diaminopimelic acid in the cell wall. It contains menaquinone MK-7 as the predominant isoprenoid quinone and the major fatty acid is anteiso-C15 : 0 (35.1 %), with a moderate amount of C16 : 0 (6.9 %). According to 16S RNA gene sequence analysis, the isolate is phylogenetically placed in the same cluster as Paenibacillus taichungensis BCRC 17757T (99.7 % nucleotide sequence identity) and Paenibacillus pabuli NBRC 13638T (99.1 %) and is closely related to Paenibacillus tundrae A10bT (98.8 %). However, phylogenetic studies based on the housekeeping gyrB gene placed A59T in a separate branch from all other related type strains. Furthermore, the results of whole genome average nucleotide identity analysis (gANI) with related type strains was lower than 91.10 % and the digital DNA-DNA hybridization value was lower than 44.30 %, which are below the threshold values for separating two species. The DNA G+C content was estimated as 46.09 mol%, based on genome sequencing. On the basis of these results, A59T represents a new species of the genus Paenibacillus, and we propose the name Paenibacillusxylanivorans sp. nov. The type strain is A59T (=DSM 107920T=NCIMB 15123T).

Diversity and distribution of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Argentina.

A total of 268 Bacillus thuringiensis strains obtained from different sources of Argentina were analyzed to determine the diversity and distribution of the cry1, cry2, cry8, cry9 and vip3A genes encoding for lepidopteran-specific insecticidal proteins. Twin strains were excluded. Ten different profiles were detected among the 80 selected B. thuringiensis strains. Two of these profiles (cry1Aa, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (35/80), and cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (25/80)) pooled 75% of the strains. The existence of this low diversity is rare, since in most of the studied collections a great diversity of insecticidal toxin gene profiles has been described. In addition, the most frequently detected profile was also most frequently derived from soil (70%), stored product dust (59%) and spider webs (50%). In contrast, the cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa profiles were mainly detected in strains isolated from leaves (40%) and dead insect larvae (50%). Six of the identified insecticidal toxin gene profiles were discovered in strains isolated from stored product dust and leaves indicating higher diversity of profiles in these kinds of sources than in others. Some strains with high insecticidal activity against Epinotia aporema (Lepidoptera) larvae were identified, which is important to explore future microbial strategies for the control of this crop pest in the region.

Selection of Bacillus thuringiensis strains toxic to cotton boll weevil (Anthonomus grandis, Coleoptera: Curculionidae) larvae.

Preliminary bioassays with whole cultures (WC) of 124 Bacillus thuringiensis strains were performed with neonate larvae of Anthonomus grandis, a major cotton pest in Argentina and other regions of the Americas. Three exotic and four native strains were selected for causing more than 50% mortality. All of them were ß-exotoxin producers. The native strains shared similar morphology of parasporal crystals, similar protein pattern and identical insecticidal gene profiles. These features resembled Lepidoptera-toxic strains. Furthermore, these strains showed a Rep-PCR pattern identical to lepidoptericidal strain HD-1, suggesting that these strains may belong to serovar kurstaki. However, some differences were observed in the plasmid profiles and in the production of ß-exotoxin. To determine the culture fractions where the insecticidal metabolites were present, bioassays including resuspended spore-crystal pellets, filtered supernatants (FS) were compared with those of WC. Both fractions tested showed some level of insecticidal activity. The results may suggest that the main toxic factors can be found in FS and could be directly correlated with the presence of ß-exotoxin. Based on the bioassays with FS and autoclaved FS, the participation of thermolabile virulence factors such as Cry1I in toxicity is neither discarded. In the selected strains, ß-exotoxin would be the major associated virulence factor; therefore, their use in biological control of A. grandis should be restricted. Nevertheless, these strains could be the source of genes (e.g., cry1Ia) to produce transgenic cotton plants resistant to this pest.

PCR-based prediction of type I ß-exotoxin production in Bacillus thuringiensis strains.

Some Bacillus thuringiensis strains secrete type I ß-exotoxin, which is a non-specific insecticidal and thermostable adenine nucleoside oligosaccharide. Toxicity bioassays and HPLC are traditional methods for detecting ß-exotoxin. With the aim of establish a first rapid approach for prediction of type I ß-exotoxin production, two PCR-based methods were successfully evaluated in B. thuringiensis strains and native isolates. In order to validate a reliable technology, results obtained by this method were correlated with that obtained from Musca domestica bioassays.

[Bacillus thuringiensis: general aspects. An approach to its use in the biological control of lepidopteran insects behaving as agricultural pests]. / Bacillus thuringiensis: generalidades. Un acercamiento a su empleo en el biocontrol de insectos lepidópteros que son plagas agrícolas.

Bacillus thuringiensis is the most widely applied biological pesticide used to control insects that affect agriculture and forestry and which transmit human and animal pathogens. During the past decades B. thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the relationships between the structure, mechanism of action, and genetics of their pesticidal crystal proteins. As a result, a coherent picture of these relationships has emerged. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins and their performance in agricultural and other natural settings. With this knowledge as background and the help of biotechnological tools, researchers are now reporting promising results in the development of more useful toxins, recombinant bacteria, new formulations and transgenic plants that express pesticidal activity, in order to assure that these products are utilized with the best efficiency and benefit. This article is an attempt to integrate all these recent developments in the study of B. thuringiensis into a context of biological control of lepidopteran insect pest of agricultural importance.

Complete Sequence and Organization of pFR260, the Bacillus thuringiensis INTA Fr7-4 Plasmid Harboring Insecticidal Genes.

We report the complete sequence and analysis of pFR260, a novel megaplasmid of 260,595 bp from the Bacillus thuringiensis strain INTA Fr7-4 isolated in Argentina. It carries 7 insecticidal genes: 3 cry8 copies previously reported, 2 vip1, and 2 vip2. Also, it carries a gene encoding a putative atypical Cry protein. These genes are arranged in a region of approximately 105 kbp in size with characteristics of a pathogenicity island with a potential coleopteran-specific insecticide profile. DNA strand composition asymmetry, as determined by GC skew analysis, and the presence of a Rep protein involved in the initiation of replication suggest a bidirectional theta mechanism of replication. In addition, many genes involved in conjugation and a CRISPR-Cas system were detected. The pFR260 sequence was deposited in GenBank under accession number KX258624.

Draft Genome Sequence of Bacillus thuringiensis INTA Fr7-4.

We report here the complete annotated 6,035,547-bp draft genome sequence of Bacillus thuringiensis INTA Fr7-4. This strain contains three cry8 and two vip1 and vip2 insecticidal toxin genes.

[Prevalence of associated microorganisms in genital discharge, Argentina]. / Prevalencia de microorganismos asociados a secrecíon genital femenina, Argentina.

OBJECTIVE: There was a significant increase in the number of women demanding gynecological care in public hospital of the Great Buenos Aires, Argentina, between 1997 and 1998. It was necessary to update the prevalence of associated microorganisms in order to review the laboratory support and adjust prevention and control guidelines. METHODS: Samples from vaginal and endocervical discharge, from total cases: 84 adolescents (15 to 19 years) and 784 adults (20-60 years) attended in 1997-1998, were studied. Neisseria gonorrhoeae, Streptococcus agalactiae, Trichomonas vaginalis, Candida spp, and bacterial vaginosis, were diagnosed applying direct detection methods and specific culture isolation. Chlamydia trachomatis (antigen detection), Ureaplasma urealyticum and Mycoplasma hominis (culture) were also studied in part of the population. RESULTS: Patient care increased steadily from 1997 and there was an increase of 2.1 times from the first semester to the last one in 1998. Bacterial vaginosis was the most prevalent disease in the adult group, with 23.8%; followed by Candida spp 17.8%; S. agalactiae 5.6%; T.vaginalis 2.4%. In 50.3% of total adult cases neither bacterial vaginosis or presence of any sought microorganisms, was detected. In the adolescents group the most frequent detection was Candida spp with a 29.7%; bacterial vaginosis in 17.8%; followed by S. agalactiae 3.6%, T.vaginalis 2.4%. Also in this group on an important number of cases, 46.4%, none bacterial vaginosis or the presence of the sought microorganism were found. In some of the adult group, C. trachomatis (7/400) 1.76%, U. urealyticum (209/340) 61.4% and M. hominis (45/272) 16.5% were detected. CONCLUSIONS: Bacterial vaginosis and Candida spp prevalence is important in both groups. The absence of N. gonorrhoeae and lower prevalence of T. vaginalis and C. trachomatis is remarkable. A high prevalence of U. urealyticum and M. hominis were also detected, but the actual pathogenic role in adult women is still under discussion. The significant increase of attendance in the public system is due to social changes in the population and not due to changes in any particular pathology. Laboratory support has to be adjusted to the most cost-effective procedures to solve the most prevalent problems.

Sequence and expression of two cry8 genes from Bacillus thuringiensis INTA Fr7-4, a native strain from Argentina.

We found and characterized two cry8 genes from the Bacillus thuringiensis strain INTA Fr7-4 isolated in Argentina. These genes, cry8Kb3 and cry8Pa3, are located in a tandem array within a 13,200-bp DNA segment sequenced from a preparation of total DNA. They encode 1,169- and 1,176-amino-acid proteins, respectively. Both genes were cloned with their promoter sequences and the proteins were expressed separately in an acrystalliferous strain of B. thuringiensis leading to the formation of ovoid crystals in the recombinant strains. The toxicity against larvae of Anthonomus grandis Bh. (Coleoptera: Curculionidae) of a spore-crystal suspension from the recombinant strain containing cry8Pa3 was similar to that of the parent strain INTA Fr7-4.

Identification, cloning and expression of an insecticide cry8 gene from Bacillus thuringiensis INTA Fr7-4.

Insecticidal activity of Bacillus thuringiensis is attributed largely to the crystal proteins. These were found with specific toxic activity against insects in different orders. A novel cry8 gene from B. thuringiensis strain INTA Fr7-4 from Argentina was characterized. The encoded protein, Cry8Qa2, is 1184 amino acids long and its sequence is more similar to those of Cry8F class. We cloned and expressed the protein in an acrystalliferous strain of B. thuringiensis using two differentially regulated promoters. The recombinant strains produced ovoid crystals with low toxicity against larvae of Anticarsia gemmatalis (Lepidoptera: Noctuidae). The morphology and insecticidal properties of these crystals resembled those produced by the native strain INTA Fr7-4.

Diversity and distribution of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Argentina / Diversidad y distribución de genes de toxinas insecticidas Lepidoptera-específicos en cepas de Bacillus thuringiensis de Argentina

A total of 268 Bacillus thuringiensis strains obtained from different sources of Argentina were analyzed to determine the diversity and distribution of the cryl, cry2, cry8, cry9 and vip3A genes encoding for lepidopteran-specific insecticidal proteins. Twin strains were excluded. Ten different profiles were detected among the 80 selected B. thuringiensis strains. Two of these profiles (cry1Aa, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (35/80), and cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (25/80)) pooled 75% of the strains. The existence of this low diversity is rare, since in most of the studied collections a great diversity of insecticidal toxin gene profiles has been described. In addition, the most frequently detected profile was also most frequently derived from soil (70%), stored product dust (59%) and spider webs (50%). In contrast, the cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa profiles were mainly detected in strains isolated from leaves (40%) and dead insect larvae (50%). Six of the identified insecticidal toxin gene profiles were discovered in strains isolated from stored product dust and leaves indicating higher diversity of profiles in these kinds of sources than in others. Some strains with high insecticidal activity against Epinotia aporema (Lepidoptera) larvae were identified, which is important to explore future microbial strategies for the control of this crop pest in the region.

Se analizaron 268 cepas de Bacillus thuringiensis obtenidas de diferentes fuentes de Argentina con el objeto de determinar la diversidad y distribución de genes cryl, cry2, cry8, cry9 y vip3A, que codifican proteínas insecticidas lepidóptero-específicas. Se excluyeron las cepas gemelas. Se detectaron solo diez perfiles diferentes entre los 80 B. thuringiensis seleccionados. Dos de estos perfiles, el cry1Aa, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa (35/80) y el cry1Aa, cry1Ab, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa (25/80), comprendieron el 75% de las cepas seleccionadas. La existencia de esta baja diversidad es una rareza, ya que en la mayor parte de las colecciones estudiadas se ha descrito una gran diversidad de perfiles de genes de toxinas insecticidas. El perfil detectado con mayor frecuencia se obtuvo principalmente de cepas procedentes de suelo (el 70% de los de esa fuente lo tenían), también fue mayoritario entre los procedentes de polvo de producto almacenado (59%) y en los que procedían de telas de arana (50%). En cambio, el perfil cry1Aa, cry1Ab, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa se detectó principalmente en las cepas aisladas de hojas (40%) y de larvas de insectos muertos (50%). Seis de los perfiles identificados fueron encontrados en cepas aisladas de polvo de producto almacenado y de hojas, lo que indica una mayor diversidad de perfiles en estas fuentes que en otras. Se identificaron algunas cepas con alta actividad insecticida contra larvas de Epinotia aporema (Lepidoptera), hallazgo importante para explorar en el futuro estrategias microbianas para el control de esta plaga en la región.

New variants of lepidoptericidal toxin genes encoding Bacillus thuringiensis Vip3Aa proteins.

Bacillus thuringiensis is an entomopathogenic bacterium characterized by producing parasporal proteinaceous insecticidal crystal inclusions during sporulation. Many strains are capable of also expressing other insecticidal proteins called Vip during the vegetative growing phase. Particularly, Vip3A proteins have activity against certain Lepidoptera species through a unique mechanism of action which emphasized their possible use in resistance management strategies against resistant pests. The aim of the work was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that can distinguish between vip3A genes from B. thuringiensis strains. In addition, 4 novel vip3Aa genes were cloned and sequenced. The method was originally based on amplification of a single PCR amplicon and the use of 2 restriction enzymes with recognition sites that facilitate simultaneous detection. Subsequently, a third restriction enzyme was used to distinguish between vip3A variants. Thirteen vip3Aa genes were identified in strains belonging to 10 different B. thuringiensis serovars. Three intra-subclass variants of vip3Aa genes could be differentiated. The presented method can serve as an invaluable tool for the investigation of known and novel vip3A genes in B. thuringiensis strains. To the best of our knowledge, this is the first report where variants of a same subclass of insecticidal genes could be distinguished following PCR-RFLP.

Selection of Bacillus thuringiensis strains toxic to cotton boll weevil (Anthonomus grandis, Coleoptera: Curculionidae) larvae / Selección de cepas de Bacillus thuringiensis tóxicas para larvas del picudo del algodonero (Anthonomus grandis, Coleoptera: Curculionidae)

Preliminary bioassays with whole cultures (WC) of 124 Bacillus thuringiensis strains were performed with neonate larvae of Anthonomus grandis, a major cotton pest in Argentina and other regions of the Americas. Three exotic and four native strains were selected for causing more than 50% mortality. All of them were β-exotoxin producers. The native strains shared similar morphology of parasporal crystals, similar protein pattern and identical insecticidal gene profiles. These features resembled Lepidoptera-toxic strains. Furthermore, these strains showed a Rep-PCR pattern identical to lepidoptericidal strain HD-1, suggesting that these strains may belong to serovar kurstaki. However, some differences were observed in the plasmid profiles and in the production of β-exotoxin. To determine the culture fractions where the insecticidal metabolites were present, bioassays including resuspended spore-crystal pellets, filtered supernatants (FS) were compared with those of WC. Both fractions tested showed some level of insecticidal activity. The results may suggest that the main toxic factors can be found in FS and could be directly correlated with the presence of β-exotoxin. Based on the bioassays with FS and autoclaved FS, the participation of thermolabile virulence factors such as Cry1I in toxicity is neither discarded. In the selected strainsβ-exotoxin would be the major associated virulence factor; therefore, their use in biological control of A. grandis should be restricted. Nevertheless, these strains could be the source of genes (e.g., crylla) to produce transgenic cotton plants resistant to this pest.

Se realizaron ensayos preliminares con cultivos completos de 124 cepas de Bacillus thuringiensis utilizando larvas neonatas de Anthonomus grandis, una plaga principal del algodón en Argentina y otras regiones de América. Se seleccionaron 3 cepas exóticas y 4 nativas por producir mortalidad superior al 50%, todas ellas productoras de β-exotoxina. Las cepas nativas presentan la misma morfología de cristales, un perfil de proteínas similar y los mismos genes insecticidas. Estas características hacen que se parezcan a cepas tóxicas para lepidópteros. Además, mostraron un perfil de Rep-PCR idéntico al de la cepa lepidoptericida HD-1, lo que indica que podrían pertenecer al serovar kurstaki. Sin embargo, se observaron diferencias en el perfil plasmídico y en la producción de β-exotoxina. Para determinar en qué fracción del cultivo se encontraban los metabolitos responsables de la toxicidad, se compararon los resultados de bioensayos en los que se utilizó biomasa, sobrenadante filtrado (SF) o cultivos completos. Ambas fracciones mostraron cierto grado de toxicidad. Los resultados indican que los principales factores tóxicos se encuentran en el sobrenadante y estarían directamente relacionados con la presencia de β-exotoxina. De acuerdo con los bioensayos de SF y SF autoclavado, no se descarta también la participación en la toxicidad de factores de virulencia termolábiles, como CrylIa. En las cepas seleccionadas, el principal factor de virulencia es la β-exotoxina, por lo que su uso debería restringirse para el control biológico de A. grandis. No obstante, estas podrían ser fuente de genes (p. ej., crylIa) para la producción de plantas de algodón transgénicas resistentes a dicha plaga.

Evidence of Bacillus thuringiensis intra-serovar diversity revealed by Bacillus cereus group-specific repetitive extragenic palindromic sequence-based PCR genomic fingerprinting.

Bacillus thuringiensis is classified into serovars on the basis of H-flagellar antigens. Several alternative typing methods have been described. Among them, a B. cereus group-specific repetitive extragenic palindromic (Rep)-PCR fingerprinting technique was shown to be discriminative and able to identify B. thuringiensis serovars. The aim of this study was to investigate the genomic diversity and relationship among B. thuringiensis strains collected from different Argentinean ecosystems. Thirty-seven B. thuringiensis reference strains and 131 Argentinean isolates were analyzed using a B. cereus group-specific Rep-PCR. Fourteen different patterns were identified among the Argentinean isolates. Eight could not be associated to any pattern obtained from a reference strain. The pattern identical to the serovar kurstaki HD-1 strain was the most frequently identified in 68 native isolates. The profiles allowed tracing a single dendrogram with two groups and eight main lineages. Some strains showed distinctive patterns despite belonging to the same serovar. An intraspecific diversity resulted from this analysis that was highlighted by this technique since strains from a given serovar showed distinct profiles. This study may help to establish a system of B. thuringiensis classification with a higher discrimination level than established by the H antigen serotyping.

Characterization of an Argentine isolate of Bacillus thuringiensis similar to the HD-1 strain.

We report the characterization of an Argentine isolate of Bacillus thuringiensis (INTA TA24-6) similar to the HD-1 strain, which harbors a cryptic cry2Ab gene that apparently is transcribed but not translated into a protein. INTA TA24-6 showed a Rep-PCR pattern identical to the HD-1 strain, a plasmid pattern that resembled that of this strain and cry1 and cry2 genes as HD-1. Screening of cry1 and cry2 genes showed that INTA TA24-6 harbors only cry1Ac and cry2Ab genes. Furthermore, crystalline inclusions of INTA TA24-6 exhibit a bipyramidal shape, typical of Lepidoptera-active B. thuringiensis strains, containing a major protein of ca. 130 kDa toxic to Epinotia aporema Wals. (Lepidoptera: Tortricidae) larvae. Neither the flat-square to cuboidal crystal nor a ca. 65 kDa protein typical of strains expressing Cry2 proteins were detected in INTA TA24-6. In agreement with this information, parasporal crystals of INTA TA24-6 did not show toxicity to Aedes aegypti L. (Diptera:Culicidae) larvae. Gene transcription analyses suggested that the cry2A gene might be cryptic in INTA TA24-6 despite its transcription at different sporulation stages.