Publications

4741

La producción de limón persa (LP) es importante para el estado de Veracruz, México. Sin embargo, se ve afectada por el Huanglongbing (HLB), causada por Candidatus Liberibacter asiaticus (CLas), un patógeno biotrófico obligado. LP presenta un cierto nivel de tolerancia al HLB, por tanto, resulta relevante estudiar su respuesta de defensa mediada por ácido salicílico (SA). Tres genes con capacidad de participar en la ruta de respuesta por SA, conocidos como NtSABP, han sido identificados en Nicotiana tabacum; sin embargo, se desconoce la presencia y actividad de dichos genes en LP en respuesta al HLB. En este trabajo se identificaron proteínas homólogas tipo SABP en el transcriptoma de LP y se determinó su grado de expresión diferencial durante la infección con CLas. Se realizó un tBLASTn en el transcriptoma de LP usando como modelo secuencias de las proteínas SABP de cinco diferentes especies, incluyendo N. tabacum. Se reconstruyó y comparó el modelo 3D de las proteínas SABP de N. tabacum y C. latifolia. Con los análisis de tBLASTn, la reconstrucción filogenética y la estructura tridimensional se logró identificar el homólogo directo de cada gen NtSABP en LP. Interesantemente, los genes ClSABP1, ClSABP2 y ClSABP3 mostraron represión en plantas infectadas con CLas. En LP hay al menos un homólogo para cada gen NtSABP. Durante la infección por CLas, estos genes se encuentran ligeramente reprimidos.

AbstractPhytoplasmas are bacteria transmitted by insects that can cause plant diseases. In Bogotá 'Candidatus Phytoplasma asteris' and ' Candidatus Phytoplasma fraxini', infect 11 species of urban trees, weeds, grass, potato and strawberry. A set of primers, that amplify both phytoplasmas species were designed and used for absolute and relative qPCR quantification of the 16SrRNA gene. The primers AJ-16Sr-F/AJ-16Sr-R allowed the amplification of ‘Ca. P. asteris’, ‘Candidatus Phytoplasma palmae’, ‘Ca. P. fraxini’ and ‘Candidatus Phytoplasma phoenicium’, not of ‘Candidatus Phytoplasma pruni’. Absolute qPCR detected phytoplasmas between 1 × 109 and 1 × 103 copies/μL DNA extract. Two species-specific hydrolysis probes, AJ-16SrI-Cy5.5 and AJ-16SrVII-TexRed, were designed to detect 'Ca. P. asteris' and 'Ca. P. fraxini' respectively, using the AJ-16Sr-F/AJ-16Sr-R primers. For relative quantification, the 18SrRNA gene was used as normalizer. Relative qPCR detected phytoplasmas between 1 × 109 and 1 × 103 copies/μL DNA extract. Multiplex reactions allowed the specific quantification of 'Ca. P. asteris', 'Ca. P. fraxini' in comparison to the normalizer. qPCR methods were validated on natural hosts Andean oak trees and leafhoppers. The relative quantification values were higher for 'Ca. P. fraxini' (x̅ RQ = 3203.1 ± 2622,9 n = 14) compared with 'Ca. P. asteris' (x̅ RQ = 14.9 ± 24,5 n = 6) in oak tree samples. In the leafhoppers, the relative quantification values ranged between RQ = 26.5 and RQ = 294,927.3 for 'Ca. P. fraxini’ and RQ = 34.8 and RQ = 1722.2 for 'Ca. P. asteris'. In conclusion, although absolute qPCR allowed the quantification of phytoplasmas by comparing Cq (quantification cycle) values of samples with a standard curve, it did not allow to differentiate between 'Ca. P. asteris' and 'Ca. P. fraxini'. In contrast, relative qPCR assays using specific hydrolysis probes allowed the specific detection and quantification of each phytoplasma, in individual and mixed infections in insect vectors and plant hosts.

Candidatus Liberibacter asiaticus (CLas) se ha dispersado rápidamente a las regiones productoras de cítricos en México, ocasionando impactos económicos y sociales diferenciados. En México, los principales avances en investigación se han centrado en el manejo del vector, Diaphorina citri y en estudios de la interacción de CLas con limón mexicano. Sin embargo, la variabilidad genética de la bacteria en regiones productoras de México se ha estudiado poco. Por lo que, en el presente estudio se analizó la variación de la población mexicana de CLas con procedimientos basados en PCR, secuenciación y electroforesis capilar a través de la detección del número de repeticiones en tándem (TRN) con dos marcadores STR, AGACACA y TACAGAA, el primero localizado en locus genómicos CLIBASIA_01645 y adenosina deaminasas. La distribución de los TRNs en la población mexicana de CLas en ambos loci mostraron variaciones con las poblaciones previamente reportadas en Florida, China y Brasil. Además, en la población mexicana se encontraron tres haplotipos, HA, HB y HC, siendo el primer reporte del haplotipo HC en las regiones citrícolas en México. Interesantemente, las subpoblaciones de CLas del Occidente y Noroeste son diferentes a la del Sureste.

The family Rhizobiaceae contains 19 validly described genera including the rhizobia groups, many of which are important nitrogen-fixing bacteria. Early classification of Rhizobiaceae relied heavily on the poorly resolved 16S rRNA genes and resulted in several taxonomic conflicts. Although several recent studies illustrated the taxonomic status of many members in the family Rhizobiaceae, several para- and polyphyletic genera still needed to be elucidated. The rapidly increasing number of genomes in Rhizobiaceae has allowed for a revision of the taxonomic identities of members in Rhizobiaceae. In this study, we performed analyses of genome-based phylogeny and phylogenomic metrics to review the relationships of 155-type strains within the family Rhizobiaceae. The UBCG and concatenated protein phylogenetic trees, constructed based on 92 core genes and concatenated alignment of 170 single-copy orthologous proteins, demonstrated that the taxonomic inconsistencies should be assigned to eight novel genera, and 22 species should be recombined. All these reclassifications were also confirmed by pairwise cpAAI values, which separated genera within the family Rhizobiaceae with a demarcation threshold of ~86%. In addition, along with the phenotypic and chemotaxonomic analyses, a novel strain BDR2-2T belonging to a novel genus of the family Rhizobiaceae was also confirmed, for which the name Ectorhizobium quercum gen. nov., sp. nov. was proposed. The type strain is BDR2-2T (=CFCC 16492T = LMG 31717T).

IntroductionNatural products discovered from bacteria provide critically needed therapeutic leads for drug discovery, and myxobacteria are an established source for metabolites with unique chemical scaffolds and biological activities. Myxobacterial genomes accommodate an exceptional number and variety of biosynthetic gene clusters (BGCs) which encode for features involved in specialized metabolism.MethodsIn this study, we describe the collection, sequencing, and genome mining of 20 myxobacteria isolated from rhizospheric soil samples collected in North America.ResultsNine isolates were determined to be novel species of myxobacteria including representatives from the genera Archangium, Myxococcus, Nannocystis, Polyangium, Pyxidicoccus, Sorangium, and Stigmatella. Growth profiles, biochemical assays, and descriptions were provided for all proposed novel species. We assess the BGC content of all isolates and observe differences between Myxococcia and Polyangiia clusters.DiscussionContinued discovery and sequencing of novel myxobacteria from the environment provide BGCs for the genome mining pipeline. Utilizing complete or near-complete genome sequences, we compare the chromosomal organization of BGCs of related myxobacteria from various genera and suggest that the spatial proximity of hybrid, modular clusters contributes to the metabolic adaptability of myxobacteria.