Publications

4359

Diaphorina citri Kuwayama (D. citri) is an insect vector of phloem-limited ‘Candidatus Liberibacter asiatus’ (CLas), the presumed pathogen of citrus Huanglongbing (HLB). Recently, our lab has preliminarily found it acquired and transmitted Citrus tristeza virus (CTV), which was previously suggested to be vectored by species of aphids. However, the influences of one of the pathogens on the acquisition and transmission efficiency of the other pathogen remain unknown. In this study, CLas and CTV acquisition and transmission by D. citri at different development stages under field and laboratory conditions were determined. CTV could be detected from the nymphs, adults, and honeydew of D. citri but not from the eggs and exuviates of them. CLas in plants might inhibit CTV acquisition by D. citri as lower CTV–positive rates and CTV titers were detected in D. citri collected from HLB-affected trees compared to those from CLas–free trees. D. citri were more likely to obtain CTV than CLas from host plants co-infected with the two pathogens. Intriguingly, CTV in D. citri facilitated the acquisition and transmission of CLas, but CLas carried by D. citri had no significant effect on the transmission of CTV by the same vector. Molecular detection and microscopy methods confirmed the enrichment of CTV in the midgut after a 72-h acquisition access period. Collectively, these results raise essential scientific questions for further research on the molecular mechanism of pathogen transmission by D. citri and provide new ideas for the comprehensive prevention and control of HLB and CTV.

El Huanglongbing (HLB), es una de las enfermedades más devastadoras de los cítricos a nivel mundial y representa una seria amenaza para la citricultura mexicana. El agente causal es una bacteria incultivable que pertenece al género Candidatus Liberibacter spp. y afecta a todas las especies de cítricos, especialmente los cítricos agrios. Para su detección, se emplean técnicas moleculares como es la reacción en cadena de la polimerasa (QT-PCR), técnicas serológicas y espectrofotométricas. El objetivo de este trabajo fue expresar la proteína recombinante BY8743 de Candidatus Liberibacter mediante electroforesis en geles de poliacrilamida al 12 % (PAGE) y Dot Blot. Para obtener la secuencia se acudió a la base de datos del National Center Biotechnology Information (NCBI). Una vez obtenida la secuencia, se revisó y modificó el marco de lectura abierto, por sus siglas en ingles Open Reading Frame (ORF) mediante el programa CLC Main Workbench 7 y se procedió a sintetizar el vector pET22b+, también se verificó el codón de inicio y de termino acoplado al vector. Para la transformación se usaron células comerciales One Shot® TOP10 Chemically Competent (InvitrogenTM), posteriormente fueron subclonadas en células de E. coli BL21. Se realizaron minipreparaciones usando un método de lisis alcalina y además se realizó una cinética de crecimiento, observando que en tres horas se alcanza la fase estacionaria. Se aplicaron concentraciones de 0.5, 0.8 y 1 mM del inductor metabólico isopropil-β-D-1-tiogalactopiranósido (IPTG) y la mejor concentración fue 1 mM, ya que a las cuatro horas después de su aplicación se pudo visualizar una proteína sobreexpresada de 38 kDa. Cuando se realizó el Dot Blot se obtuvieron resultados sobresalientes de inmunoexpresión en las tres concentraciones del inductor utilizado a diferencia del control y de las células sin transfectar.

El Huanglongbing (HLB), es una de las enfermedades más devastadoras de los cítricos a nivel mundial, el agente causal es una bacteria del género Candidatus Liberibacter spp., y afecta a todas las especies de cítricos, especialmente los cítricos agrios. Actualmente se han confirmado tres especies de la bacteria: Ca. L. asiáticus, Ca. L. americanus y Ca. L. africanus. Esta bacteria es transmitida por dos insectos vectores: Diaphorina citri (América y Asia) y Trioza erytrae (África), lo que favorece su rápida y eficaz diseminación, ocasionando cuantiosas pérdidas económicas, así como problemas sociales y ambientales. En este trabajo se realizó la optimización de la Reacción en Cadena de la Polimerasa punto final y Tiempo Real (PCR punto final y PCR-TR) para la detección de HLB en naranjo agrio en la zona norte del estado de Tamaulipas, México. Se procesaron 178 muestras (168 fueron de nervadura central de la hoja, ocho de raíz y dos muestras del vector). Mediante PCR punto final se detectó a la bacteria a partir del DNA extraído de hoja, pero no se detectó en el DNA extraído a partir de raíz y del vector. Mediante la PCR-TR se detectó a la bacteria a partir del ADN extraído de hoja y raíz de naranjo agrio y del insecto vector. Estos resultados demuestran que la PCR-TR es más eficiente que la PCR punto final para la detección de la bacteria en diferentes tejidos de la planta de naranjo agrio y en el insecto vector.

The citrus industry in Florida faces a destructive endemic disease, known as huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium, and transmitted by the Asian citrus psyllid (ACP). Rootstocks are regarded as critical to keep citrus production commercially viable and help trees cope with the disease. Although most scions are susceptible, some rootstocks are HLB-tolerant and may influence ACP infestation and CLas colonization and therefore the grafted tree tolerance. This study aimed to elucidate the relative influence of rootstock and scion on insect vector infestation and CLas colonization under natural HLB-endemic conditions. Seven commercial rootstock cultivars with different genetic backgrounds were grafted with ‘Valencia’ sweet orange (Citrus sinensis) or were self-grafted (non-‘Valencia’) and planted in an open field where ACP and CLas were abundant. ACP infestation was determined weekly during periods of leaf flushing, and leaves and roots were analyzed every 3 months to determine CLas titers. Trees with ‘Valencia’ scion were more attractive to the psyllids than non-‘Valencia’ scions. This was also associated with a higher number of bacteria and a larger abundance of foliar HLB symptoms. The influence of the rootstock on the psyllid attraction of grafted ‘Valencia’ scion was less evident, and leaf CLas titers were similar regardless of the rootstock. Among the non-‘Valencia’ scions, Carrizo had the lowest and US-942 the highest leaf CLas titers. Root CLas titers also varied among cultivars, and standard sour orange roots harbored more bacteria than some trifoliate orange hybrid rootstocks such as US-942. In some trees, CLas was detected first in the roots 4 months after planting, but root CLas titers remained low throughout the study. In contrast, leaf CLas titers increased over time and were considerably higher than root titers from 7 months until the end of the study, 15 months after planting. Overall, the results of this study demonstrate a greater relative influence of the scion than the rootstock on ACP infestation and CLas colonization during the early stages of infection. This suggests that other cultivar-specific traits, such as the ability to tolerate other stresses and to absorb water and nutrients more efficiently, along with influences on the scion phenology, may play a larger role in the rootstock influence on the grafted tree tolerance during the later stages of HLB progression.

Abstract Cable bacteria of the Desulfobulbaceae family are centimeter-long filamentous bacteria, which are capable of conducting long-distance electron transfer. Currently, all cable bacteria are classified into two candidate genera: Candidatus Electronema, typically found in freshwater environments, and Candidatus Electrothrix, typically found in saltwater environments. This taxonomic framework is based on both 16S rRNA gene sequences and metagenome-assembled genome (MAG) phylogenies. However, most of the currently available MAGs are highly fragmented, incomplete, and thus likely miss key genes essential for deciphering the physiology of cable bacteria. Also, a closed, circular genome of cable bacteria has not been published yet. To address this, we performed Nanopore long-read and Illumina short-read shotgun sequencing of selected environmental samples and a single-strain enrichment of Ca. Electronema aureum. We recovered multiple cable bacteria MAGs, including two circular and one single-contig. Phylogenomic analysis, also confirmed by 16S rRNA gene-based phylogeny, classified one circular MAG and the single-contig MAG as novel species of cable bacteria, which we propose to name Ca. Electronema halotolerans and Ca. Electrothrix laxa, respectively. The Ca. Electronema halotolerans, despite belonging to the previously recognized freshwater genus of cable bacteria, was retrieved from brackish-water sediment. Metabolic predictions showed several adaptations to a high salinity environment, similar to the “saltwater” Ca. Electrothrix species, indicating how Ca. Electronema halotolerans may be the evolutionary link between marine and freshwater cable bacteria lineages.

Abstract Citrus huanglongbing (HLB), associated with the unculturable phloem‐limited bacterium “ Candidatus Liberibacter asiaticus” ( C Las), is the most devastating disease in the citrus industry worldwide. However, the pathogenicity of C Las remains poorly understood. In this study, we show that AGH17488, a secreted protein encoded by the prophage region of the C Las genome, suppresses plant immunity via targeting the host ASCORBATE PEROXIDASE6 (APX6) protein in Nicotiana benthamiana and Citrus sinensis . The transient expression of AGH17488 reduced the chloroplast localization of APX6 and its enzyme activity, inhibited the accumulation of reactive oxygen species (H 2 O 2 and O 2 − ) and the lipid oxidation endproduct malondialdehyde in plants, and promoted the proliferation of Pseudomonas syringae pv. tomato DC3000 and Xanthomonas citri subsp. citri . This study reveals a novel mechanism underlying how C Las uses a prophage‐encoded effector, AGH17488, to target a reactive oxygen species accumulation‐related gene, APX6 , in the host to facilitate its infection.