Publications

4364

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 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.

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.

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.

AbstractIncreasing evidence is proving the biological significance of the phytoplasma-secreted proteins. However, besides a few Sec-dependent secretory proteins, no other phytoplasma-secreted proteins have been reported yet. Candidatus Phytoplasma ziziphi is a phytoplasma that causes witches’-broom, a devastating jujube disease prevalent in east Asia. In this study, using the SecretomeP server coupled with an Escherichia coli-based alkaline phosphatase assay, we identified 25 non-classically secreted proteins (ncSecPs) from Ca. P. ziziphi, a novel type of secreted protein associated with phytoplasmas. Among them, six were characterized as hypersensitive cell death response (HR) suppressors that significantly attenuated both Bax- and INF1-triggered HR and H2O2 accumulation in Nicotiana benthamiana, indicating a so-far unknown role of the phytoplasma-secreted proteins. Further, we demonstrated that despite the diverse subcellular localizations in the N. benthamiana cells, the six HR-suppressing ncSecPs enhanced the gene expression of several known cell death inhibitors, including pathogenesis-related proteins (NbPR-1, NbPR-2, and NbPR-5) and Bax inhibitor-1 (NbBI-1 and NbBI-2). Together, our data indicated that Ca. P. ziziphi has evolved an arsenal of ncSecPs that jointly circumvent HR by activating the plant cell death inhibitors, thus providing new insight into understanding the pathogenesis of phytoplasmas.

Abstract Background Salicylic Acid (SA) is a pivotal phytohormone in plant innate immunity enhancement of triggered by various pathogens, such as Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (HLB). WRKY is a plant specific transcription factor (TF) family, which plays crucial roles in plant response to biotic stresses. So far, the evolutionary history, functions, and expression patterns under SA treatment and CLas infection of WRKY family are poorly understood in Citrus, despite the release of the genome of several Citrus species. A comprehensive genomic and expressional analysis is worth to conduct for this family. Results Here, a genome-wide identification of WRKY TFs was performed in two Citrus species: Citrus sinensis (HLB-sensitive) and Poncirus trifoliata (HLB-tolerant). In total, 52 CsWRKYs and 51 PtrWRKYs were identified, whose physical and chemical properties, chromosome locations, phylogenetic relationships and structural characteristics were comparatively analyzed. Especially, expression patterns of these WRKY genes before and after SA treatment and CLas infection were compared. Based on this result, seven pairs of orthologous WRKY genes showing opposite expression patterns in two Citrus species were screened out. Moreover, two pairs of orthologous WRKY genes with significant differences in the number or type of stress-responsive cis-elements in the promoter regions were discovered. Subcellular localization and transcriptional activation activity assays revealed that these two pairs of orthologous genes are classic WRKY TFs localize in the nucleus and could function as transcriptional activators. Conclusion In this study, we systematically analyzed the genomic characterization of WRKY family in two Citrus species, together with the analyses of expression patterns under SA signaling and CLas infection. Our study laid a foundation for further study on the function of WRKY TFs in HLB response and SA signaling of Citrus.

Production of organic molecules is largely depending on fossil fuels. A sustainable alternative would be the synthesis of these compounds from CO2 and a cheap energy source, such as H2, CH4, NH3, CO, sulfur compounds or iron(II). Volcanic and geothermal areas are rich in CO2 and reduced inorganic gasses and therefore habitats where novel chemolithoautotrophic microorganisms for the synthesis of organic compounds could be discovered. Here we describe “Candidatus Hydrogenisulfobacillus filiaventi” R50 gen. nov., sp. nov., a thermoacidophilic, autotrophic H2-oxidizing microorganism, that fixed CO2 and excreted no less than 0.54 mol organic carbon per mole fixed CO2. Extensive metabolomics and NMR analyses revealed that Val, Ala and Ile are the most dominant form of excreted organic carbon while the aromatic amino acids Tyr and Phe, and Glu and Lys were present at much lower concentrations. In addition to these proteinogenic amino acids, the excreted carbon consisted of homoserine lactone, homoserine and an unidentified amino acid. The biological role of the excretion remains uncertain. In the laboratory, we noticed the production under high growth rates (0.034 h−1, doubling time of 20 h) in combination with O2-limitation, which will most likely not occur in the natural habitat of this strain. Nevertheless, this large production of extracellular organic molecules from CO2 may open possibilities to use chemolithoautotrophic microorganisms for the sustainable production of important biomolecules.