Jorge E. Ibarra

Anastrepha ludens (Loew) (Diptera: Tephritidae) is a major pest of mango and citrus in Mexico. It is usually controlled by applying several strategies in a holistic approach. The entomopathogenic fungus Beauveria bassiana (Bals.-Criv.) Vuill. (Cordycipitaceae) is a biocontrol agent that infects adults of many tephritids pests including A. ludens. In this work, we carried out 4 experiments with the objective of estimating the mortality of A. ludens adults when different conidial concentrations of the fungus B. bassiana were applied to the soil at different humidity, texture, and temperature conditions under controlled laboratory conditions. In a first experiment, mortality ranged from 38.3 to 79.8%, with the highest mortality occurring at a concentration of 800 mg of formulated conidia per kg of soil, while the lowest mortality was recorded at a concentration of 400 mg/kg. In a second experiment, when soils with different texture were investigated at 12% humidity, the pathogenicity of B. bassiana on A. ludens adults was similar in sandy clay soil and sandy loam (64.0% mortality), but was lower in sandy soil with 36.0% mortality. In a third experiment on humidity tests, fly mortality of A. ludens adults ranged from 43.0 to 79.8%, showing the highest mortality at 12% soil humidity and the lowest at 21%. Mortality was similar between 15 to 35 °C (>83%). Emergence of A. ludens adults in the controls ranged between 94.5 and 98.3%. The soil humidity was the major factor that significantly affected fungal efficacy. The study demonstrated the potential of using B. bassiana applied to the soil for management of A. ludens and other tephritids. However, B. bassiana must be evaluated under field conditions before recommendations can be made. Anastrepha ludens (Loew) (Diptera: Tephritidae) es la principal plaga del mango y cítricos en México. Actualmente para su manejo se aplican varias estrategias de control con un enfoque holístico. El hongo entomopatógeno, Beauveria bassiana (Bals.-Criv.) Vuill. (Cordycipitaceae) es un agente de control biológico que ataca adultos de muchas tefrítidos que son plagas, incluyendo A. ludens. En este trabajo, realizamos cuatro experimentos y el objetivo fue estimar la mortalidad de adultos de A. ludens con diferentes concentraciones de conidios del hongo B. bassiana, que fueron aplicados en suelo con diferentes humedades, texturas y temperatura, en condiciones controladas de laboratorio. En el primer experimento, la mortalidad varió de 38.3 a 79.8%, ocurriendo la mortalidad más alta a la concentración de 800 mg del formulado de conidios/kg de suelo, mientras que la mortalidad más baja se registró en la concentración de 400 mg/kg de suelo. En el segundo experimento, cuando se evaluaron diferentes texturas de suelo con 12% de humedad, la patogenicidad de B. bassiana sobre adultos de A. ludens fue similar en suelo areno-arcilloso y areno-limoso (64.0% de mortalidad), pero fue más bajo en suelo arenoso donde hubo 36.0% de mortalidad. En el tercer experimento, cuando se evaluó la humedad del suelo, la mortalidad de adultos de A. ludens varió de 43.0 a 79.8%, donde la mayor mortalidad se registró en suelo con 12% de humedad y la menor mortalidad fue en suelo con 21% de humedad. En el cuarto experimento, la mortalidad fue similar cuando la temperatura del suelo varió de 15 a 35 °C, siendo siempre >83%. La emergencia de adultos en los testigos varió de 94.5 a 98.3%. La humedad del suelo fue el mayor factor que afectó significativamente la eficacia del hongo. En este estudio se demostró el potencial del hongo B. bassiana aplicado al suelo para el control de A. ludens y otros tefrítidos. Sin embargo, es necesario que B. bassiana sea evaluado bajo condiciones de campo antes de recomendar su uso.

To demonstrate the ability of Bacillus thuringiensis to penetrate as spore-crystal complex to the internal tissues of bean plants, and keep its insecticidal activity. To test the vertical transmission of the spore-crystal complex in Arabidopsis thaliana. The experimental strain was transformed with the pMUTIN-gfp plasmid which labelled the spores of B. thuringiensis HD-73 with the GFP protein. Once the rhizosphere of the bean plants was inoculated with the labelled strain, the bacterium was recovered from leaves, stems, and petioles. Furthermore, toxicity of treated plants was significantly higher than control plants when bio-assayed on cabbage looper larvae. The labelled strain was recovered from the dead insects. When the rhizosphere of A. thaliana plants was inoculated with the labelled strain, mature seeds from these plants were surface-sterilized and grown under in vitro conditions. The labelled strain was recovered from the seedlings. We showed that B. thuringiensis subsp. kurstaki (HD-73) in the rhizosphere can translocate to upper tissues of bean plants, and keep its insecticidal activity. Transmission of the labelled B. thuringiensis strain passed to the next generation of A. thaliana. The role of B. thuringiensis as a potential facultative endophyte bacterium and the possible biotechnological repercussions are discussed.

The LBIT-1200 strain of Bacillus thuringiensis was recently isolated from soil, and showed a 6.4 and 9.5 increase in toxicity, against Manduca sexta and Trichoplusia ni, respectively, compared to HD-73. However, LBIT-1200 was still highly similar to HD-73, including the production of bipyramidal crystals containing only one protein of ∼130 000 kDa, its flagellin gene sequence related to the kurstaki serotype, plasmid and RepPCR patterns similar to HD-73, no production of β-exotoxin and no presence of VIP genes. Sequencing of its cry gene showed the presence of a cry1Ac-type gene with four amino acid differences, including two amino acid replacements in domain III, compared to Cry1Ac1, which may explain its higher toxicity. In conclusion, the LBIT-1200 strain is a variant of the HD-73 strain but shows a much higher toxicity, which makes this new strain an important candidate to be developed as a bioinsecticide, once it passes other tests, throughout its biotechnological development.

Six strains of Bacillus thuringiensis previously selected as highly toxic against Manduca sexta and Plutella xylostella were analyzed by PCR screening in order to identify the cry genes active on Lepidoptera. According to their gene content and insecticidal potency, these strains were cultured and aliquots taken at different pre- and post-sporulation times. Total RNA was extracted and used as template in RT-PCR analyses directed to identify mRNAs of the previously identified cry genes. Results showed transcription of genes cry1A, cry1E, cry1I, and cry2 even before the onset of sporulation. However, this early transcription did not lead to an appreciable parasporal protein synthesis until t₅–t₉, as deduced from SDS-PAGE profiles. As for cry1I gene, the corresponding protein was not detected, as expected, but cry1I mRNAs were present at least until t₅. Interestingly, strains expressing four cry genes from the end of the log phase onwards exhibited kinetics characterized by a very long transition phase, whereas the strain expressing only one cry gene showed a very short transition phase. Strains expressing three genes showed an intermediate profile. These results indicate that the transcription of B. thuringiensis cry1 and cry2 genes in natural strains can start several hours before massive crystal synthesis occurs and that this translation is probably competing with transcriptional regulators required for the sporulation onset.

On the basis of the known cry gene sequences of Bacillus thuringiensis , three sets of primers were designed from four conserved blocks found in the delta-endotoxin-coding region. The primer pairs designed amplify the regions between blocks 1 and 5, 2 and 5, and 1 and 4. In silico analyses indicated that 100% of the known three-domain cry gene sequences can be amplified by these sets of primers. To test their ability to amplify known and unknown cry gene sequences, 27 strains from the CINVESTAV (LBIT series) collection showing atypical crystal morphology were selected. Their DNA was used as the template with the new primer system, and after a systematic amplification and sequencing of the amplicons, each strain showed one or more cry -related sequences, totaling 54 different sequences harbored by the 27 strains. Seven sequences were selected on the basis of their low level of identity to the known cry sequences, and once cloning and sequencing of the complete open reading frames were done, three new cry -type genes (primary ranks) were identified and the toxins that they encode were designated Cry57Aa1, Cry58Aa1, and Cry59Aa1 by the B. thuringiensis Toxin Nomenclature Committee. The rest of the seven sequences were classified Cry8Ka2, Cry8-like, Cry20Ba1, and Cry1Ma1 by the committee. The crystal morphology of the selected strains and analysis of the new Cry protein sequences showed interesting peculiarities.

Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-μm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.

Practically all Bacillus thuringiensis strains contain a set of self-replicating, extrachromosomal DNA molecules or plasmids, which vary in number and size in the different strains. The plasmid patterns obtained from gel electrophoresis have previously been used as a tool to characterize strains, but comparison of the plasmid patterns has been limited in the number and diversity of strains analyzed. In this report, we were able to compare the plasmid patterns of 83 type strains (out of 84) and 47 additional strains from six serotypes. The information obtained from this comparison showed the importance of this tool as a strain characterization procedure and indicates the complexity and uniqueness of this feature. For example, with one exception, all type strains showed a unique plasmid pattern. All were unique in such a way that none showed even a single comigrating plasmid in the agarose gels, and therefore, cluster analysis was impossible, indicating that plasmid patterns are qualitative rather than quantitative features. Furthermore, comparison between strains belonging to the same serotype showed a great difference in variability. Some serotypes (e.g., israelensis) showed the same basic pattern among all its strains, while other serotypes (e.g., morrisoni) showed a great diversity of patterns. These results indicate that plasmid patterns are valuable tools to discriminate strains below the serotype level.

In this review we cover the biological control of insects, bacteria and fungus that affect different crops. Using different microorganism as bacteria viruses and fungus can do the biological control of these important problems. In this work we describe with detail the mode of action of the different microorganisms used to control insects and plant diseases. We also present novel strategies to improve the efficiency of these microorganisms against their targets and we present the development and production of several formulations to be used in the fields for the biological control of some plant problems.

A total of 119 Bacillus thuringiensis strains (83 type strains and 26 native isolates), as well as five B. cereus group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six B. cereus group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive B. cereus (Bc)-Rep-PCR pattern, except for B. thuringiensis serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native β-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the B. thuringiensis type strain. The B. cereus group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including nonflagellar and self-agglutinated strains.