Publications

3825

The Asian citrus psyllid (ACP) is an important vector of the HLB pathogen, which is a major threat to citrus production around the world. Bacterial communities harbored by insects could be affected by different environmental factors.

Phytoplasmas are dangerous bacteria severely infecting agricultural production worldwide. In the present study, the identification of phytoplasmas infecting tomato plants showing symptoms such as small leaves, flower abnormalities, stunting, witches' broom, and reddening was performed. Five plants, two symptomatic and three asymptomatic, were tested to verify phytoplasma infection. Total DNA isolated from 5 leaf samples was used as a template for PCR reactions. The phytoplasma agents were confirmed in the two symptomatic samples. BLASTn search of 16S rRNA of two sequences shared identity similarity of 99.84% with ‘Candidatus Phytoplasma australasia’. Computer-simulated virtual RFLP profiles show that the 16S rRNA sequences is identical to the reference pattern of the 16SrII-D subgroup, with a similarity coefficient of 1.00. Based on BLAST, virtual RFLP, and phylogenetic dendrogram, the identified phytoplasma strains are enclosed in the 16SrII-D subgroup. This is the first report of tomato witches' broom disease related to 16SrII-D subgroup phytoplasma strains in the Antalya province of Türkiye.

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.

Citrus Huanglongbing (HLB) is the most devastating disease of citrus, presumably caused by “Candidatus Liberibacter asiaticus” (CaLas). Although transcriptomic profiling of HLB-affected citrus plants has been studied extensively, the initial steps in pathogenesis have not been fully understood. In this study, RNA sequencing (RNA-seq) was used to compare very early transcriptional changes in the response of Valencia sweet orange (VAL) to CaLas after being fed by the vector, Diaphorina citri (Asian citrus psyllid, or ACP). The results suggest the existence of a delayed defense reaction against the infective vector in VAL, while the attack by the healthy vector prompted immediate and substantial transcriptomic changes that led to the rapid erection of active defenses. Moreover, in the presence of CaLas-infected psyllids, several downregulated differentially expressed genes (DEGs) were identified on the pathways, such as signaling, transcription factor, hormone, defense, and photosynthesis-related pathways at 1 day post-infestation (dpi). Surprisingly, a burst of DEGs (6,055) was detected at 5 dpi, including both upregulated and downregulated DEGs on the defense-related and secondary metabolic pathways, and severely downregulated DEGs on the photosynthesis-related pathways. Very interestingly, a significant number of those downregulated DEGs required ATP binding for the activation of phosphate as substrate; meanwhile, abundant highly upregulated DEGs were detected on the ATP biosynthetic and glycolytic pathways. These findings highlight the energy requirement of CaLas virulence processes. The emerging picture is that CaLas not only employs virulence strategies to subvert the host cell immunity, but the fast-replicating CaLas also actively rewires host cellular metabolic pathways to obtain the necessary energy and molecular building blocks to support virulence and the replication process. Taken together, the very early response of citrus to the CaLas, vectored by infective ACP, was evaluated for the first time, thus allowing the changes in gene expression relating to the primary mechanisms of susceptibility and host–pathogen interactions to be studied, and without the secondary effects caused by the development of complex whole plant symptoms.

Este trabajo se realizó con el objetivo de expresar el péptido de membrana externa C6XFB8 de Candidatus Liberibacter asiaticus (CLas), cepa psy62 y su detección mediante electroforesis en geles de poliacrilamida al 12 %, bajo condiciones desnaturalizantes (PAGE-SDS). La secuencia de CLas, se obtuvo en la base de datos del National Center Biotechnology International (NCBI). El marco de lectura abierto, por sus siglas en ingles Open Reading Frame (ORF), fue revisado y modificado. Mediante el programa CLC Main Workbench 7, se verificó que el codón de inicio y término estuviera acoplado al vector y se sintetizó el vector pET22b+. La transformación genética se realizó usando células One Shot TOP10 químicamente competentes, y se hicieron minipreparaciones de las clonas transformadas mediante el método de lisis alcalina. El ADN plasmídico fue subclonado en células BL21 (DE3), con las clonas obtenidas se realizó una cinética de crecimiento para conocer en qué momento y a que concentración se debía aplicar el inductor metabólico isopropil β-D-1 tiogalactopiranósido (IPTG) y de esta manera expresar la proteína C6XFB8 en la mayor cantidad posible. Se obtuvo la inducción de la expresión de la proteína y se detectó su presencia mediante análisis en SDS-PAGE y Western Blot. La mejor concentración del inductor para expresar el péptido recombinante del gen CLIBASIA 02425 de Candidatus Liberibacter asiaticus bajo el promotor T7lac fue de 0.8 mM.

‘Candidatus Liberibacter solanacearum’ contains two solanaceous crop-infecting haplotypes, A and B. Two haplotype A draft genomes were assembled and compared with ZC1 (haplotype B), revealing inversion and relocation genomic rearrangements, numerous single-nucleotide polymorphisms, and differences in phage-related regions. Differences in prophage location and sequence were seen both within and between haplotype comparisons. OrthoMCL and BLAST analyses identified 46 putative coding sequences present in haplotype A that were not present in haplotype B. Thirty-eight of these loci were not found in sequences from other Liberibacter spp. Quantitative polymerase chain reaction (qPCR) assays designed to amplify sequences from 15 of these loci were screened against a panel of ‘Ca. L. solanacearum’-positive samples to investigate genetic diversity. Seven of the assays demonstrated within-haplotype diversity; five failed to amplify loci in at least one haplotype A sample while three assays produced amplicons from some haplotype B samples. Eight of the loci assays showed consistent A–B differentiation. Differences in genome arrangements, prophage, and qPCR results suggesting locus diversity within the haplotypes provide more evidence for genetic complexity in this emerging bacterial species.