Sobrevivencia de micelio y genes asociados a la germinación no errumpente de Sclerotium cepivorum Berk

Se determinó el tiempo que el micelio de Sclerotium cepivorum Berk. puede permanecer viable tanto en condiciones de estrés por falta de humedad in vitro, como en suelo en ausencia del hospedante. El micelio permaneció viable hasta por 123 días en cajas Petri conteniendo papa-dextrosa-agar visiblemente seco. En suelo tratado a 15 lb de presión y 120°C por 4h, en dos días consecutivos y en el tratado a 2.5 lb durante 20 min a 65°C, una sola vez, los fragmentos de micelio de S. cepivorum permanecieron viables hasta 12 y 10 días posteriores a la incorporación del mismo al suelo, respectivamente; en contraste con suelo no esterilizado ya no se detectó la presencia del patógeno en este lapso. Los resultados sugieren que el micelio de S. cepivorum posee la capacidad de sobrevivir bajo estrés por falta de humedad en condiciones in vitro, en suelo no esterilizado es incapaz de competir con la biota del suelo. Se generó una biblioteca sustractiva con los genes asociados a la germinación no errumpente de Sclerotium cepivorum. Para generar la biblioteca sustractiva se determinaron las condiciones óptimas para obtener germinación errumpente y no errumpente de esclerocios de S. cepivorum. A 22º C, el 96.7% de los esclerocios germinaron de forma no errumpente en presencia de agua, y el 91.67% de los esclerocios germinó de forma errumpente en presencia de disulfuro de alilo (DAS) al 2%. En la biblioteca sustractiva se obtuvieron 23 fragmentos de genes asociados a la germinación no errumpente de S. cepivorum. Estos genes tuvieron similitudes significativas con 15 proteínas diferentes, las cuales participan en los procesos de respiración, transcripción, traducción, transducción de señales y síntesis de aminoácidos y proteínas. Los resultados sugieren que la germinación no errumpente es más rápida que la germinación errumpente debido a un incremento en los procesos metabólicos de los esclerocios de S. cepivorum germinados de forma no errumpente.________The time that mycelium of Sclerotium cepivorum Berk. can remain viable under stress by the lack of moisture both in vitro conditions and in soil in absence of the host, was determined. Mycelium remained viable by 123 days in Petri dishes containing potato-dextrose-agar completely dehydrated. In soil treated at 15 lb pressure and 120°C for 4h, in two consecutive days (double sterilization) and in soil treated to 2.5 lb for 20 min at 65°C once (single sterilization) the fragments of mycelia of Sclerotium cepivorum Berk. remained viable for 12 and 10 days after the incorporation of the mycelia to the soil, respectively; but in non-sterilized soil the pathogen was not detected in this lapse. The results suggest that the mycelium of Sclerotium cepivorum Berk. has the capacity to survive under moisture stress in vitro conditions, but in non-sterilized soil apparently it was unable to compete with the soil biota. A subtractive library of genes associated to hyphal type of Sclerotium cepivorum was generated. In order to generate the subtractive library, optimum conditions to have eruptive and hyphal germination of S. cepivorum sclerotia were determined. At 22º C, 96.7% of sclerotia germinated hyphally in water presence and 91.67% of sclerotia germinated eruptively in presence of 2% allyl disulfide (DAS). 23 fragments of genes associated to hyphal germination of S. cepivorum were obtained in the subtractive library. Those genes had significant similarity to 15 different proteins that participate in respiration, transcription, translate, signal transduction and amino acid and protein synthesis process. The results suggest that hyphal germination is faster than eruptive germination because have an increment in the metabolic process in sclerotia of S. cepivorum germinated hyphally.Tesis (Doctorado en Ciencias, especialista en Entomología y Acarología).- Colegio de Postgraduados, 2009.CONACY

Bacterial communities of the psyllid pest Bactericera cockerelli (Hemiptera: Triozidae) Central haplotype of tomato crops cultivated at different locations of Mexico

Background The psyllid, Bactericera cockerelli, is an insect vector of ‘Candidatus Liberibacter’ causing “Zebra chip” disease that affects potato and other Solanaceae crops worldwide. In the present study, we analyzed the bacterial communities associated with the insect vector Bactericera cockerelli central haplotype of tomato crop fields in four regions from Mexico. Methods PCR was used to amplify the mitochondrial cytochrome oxidase I gene (mtCOI) and then analyze the single nucleotide polymorphisms (SNP) and phylogenetic analysis for haplotype identification of the isolated B. cockerelli. Moreover, we carried out the microbial diversity analysis of several B. cockerelli collected from four regions of Mexico through the NGS sequencing of 16S rRNA V3 region. Finally, Wolbachia was detected by the wsp gene PCR amplification, which is the B. cockerelli facultative symbiont. Also we were able to confirm the relationship with several Wolbachia strains by phylogenetic analysis. Results Our results pointed that B. cockerelli collected in the four locations from Mexico (Central Mexico: Queretaro, and Northern Mexico: Sinaloa, Coahuila, and Nuevo Leon) were identified, such as the central haplotype. Analyses of the parameters of the composition, relative abundance, and diversity (Shannon index: 1.328 ± 0.472; Simpson index 0.582 ± 0.167), showing a notably relatively few microbial species in B. cockerelli. Analyses identified various facultative symbionts, particularly the Wolbachia (Rickettsiales: Anaplasmataceae) with a relative abundance higher. In contrast, the genera of Sodalis and ‘Candidatus Carsonella’ (Gammaproteobacteria: Oceanospirillales: Halomonadaceae) were identified with a relatively low abundance. On the other hand, the relative abundance for the genus ‘Candidatus Liberibacter’ was higher only for some of the locations analyzed. PCR amplification of a fragment of the gene encoding a surface protein (wsp) of Wolbachia and phylogenetic analysis corroborated the presence of this bacterium in the central haplotype. Beta-diversity analysis revealed that the presence of the genus ‘Candidatus Liberibacter’ influences the microbiota structure of this psyllid species. Conclusions Our data support that the members with the highest representation in microbial community of B. cockerelli central haplotype, comprise their obligate symbiont, Carsonella, and facultative symbionts. We also found evidence that among the factors analyzed, the presence of the plant pathogen affects the structure and composition of the bacterial community associated with B. cockerelli

Candidatus Liberibacter solanacearum patógeno vascular de solanáceas: Diagnóstico y control

Candidatus Liberibacter solanacearum is a Gram-negative bacterium, obligate parasite of the phloem in Solanaceus crops, not cultivable in vitro. It is transmitted vertical and horizontally by Bactericera cockerelli. In Mexico, CLso is a pathogen asociated with the diseases "permanente del tomate", "punta morada de la papa" (Zebra chip) and "variegado del chile". The symptoms caused by CLso depend on the farming and the stage of growth of the host but it mainly consists in yellowing and deformation of the leaf blade, due to the feeding of the vector and the colonization of the pathogen. Infections caused by CLso reduce the quality and the commercial value of the product in the market. The presence of this bacterium has been detected in the states of Coahuila, Sinaloa and Guanajuato Mexico through molecular techniques, the control of the diseases is focused on the control of the vector, by means of cultural practices and the application of chemical and biological agents. Therefore, the objective of this work is to stand out the current situation of the distribution of CLso in Mexico, the diagnostic methods and the strategies for the integrated management of the disease and the vector.Candidatus Liberibacter solanacearum (CLso) es una bacteria fitopatógena Gram-negativa, limitada al floema en solanáceas y no cultivable in vitro. Es transmitida de manera vertical y horizontal por el psílido Bactericera cockerelli. En México se asocia como responsable de la enfermedad "permanente del tomate", "punta morada de la papa" (Zebra chip) y "variegado del chile". Los síntomas causados por la bacteria varían según el cultivar y la etapa de crecimiento del hospedante pero consisten principalmente en amarillamientos y deformación de la lámina foliar, debido a la alimentación del vector y la colonización del patógeno. Las infecciones ocasionadas por CLso reducen la calidad del producto y el valor comercial en el mercado. La presencia de esta bacteria ha sido detectada en los estados de Coahuila, Sinaloa y Guanajuato, México a través de técnicas moleculares; mientras que el control de la enfermedad se encuentra enfocado en el vector, mediante prácticas culturales y la aplicación de agentes químicos y biológicos. Por lo anterior el objetivo del trabajo es puntualizar la situación actual de la distribución de CLso en México, los métodos de diagnóstico y las estrategias para el manejo integrado de la enfermedad y el vector