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El Huanglongbing (HLB) es una enfermedad considerada como la más destructiva para los cítricos en el mundo, es causada por Candidatus Liberibacter asiaticus (CLas) y diseminada por el vector Diaphorina citri. El HLB ha generado cuantiosas pérdidas económicas en la citricultura mundial. Todas las variedades comerciales de cítricos son susceptibles a esta enfermedad. La naturaleza de CLas como parásito intra celular estricto limitado al floema dificulta el estudio fitopatológico de la enfermedad. Las herramientas “ómicas”, que permiten aplicar análisis comparativos con enfoque masivos, han resultado útiles para describir la interacción entre el agente patogénico y diferentes especies de cítricos, generando conocimiento sobre las bases moleculares de la patogenicidad de CLas y de las respuestas de los hospederos ante la infección. Sin embargo, muchos procesos inmersos en la compleja interacción CLas-cítricos aún no son del todo comprendidos. En la presente revisión se resumen algunos de los principales hallazgos en la última década relacionados con los posibles mecanismos de patogenicidad de CLas a nivel molecular, así como de las respuestas transcripcionales y metabólicas inducidas en cítricos tolerantes o susceptibles, ante la infección. Este conocimiento es necesario para el diseño e implementación de nuevas estrategias para el manejo sustentable de la enfermedad.

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).

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, ‘<i>Candidatus</i> Liberibacter asiaticus’ (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, <i>Diaphorina citri</i> Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy <i>nrd</i>B gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the <i>nrd</i>B target as low as ~2.6 Log<sub>10</sub> copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.