Publications

4785

The Asian citrus psyllid, Diaphorina citri, transmits the putative causal agent of citrus greening disease, ‘ Candidatus Liberibacter asiaticus’. The transmission occurs in a propagative, circulative manner. Specific protein–protein interactions are required for the accomplishment of the transmission process. We employed the far-Western technique, also known as protein overlay assay, and mass spectrometry to identify D. citri proteins that specifically recognize membrane proteins of ‘ Ca. L. asiaticus’. Whole-body protein extracts of D. citri were separated by electrophoresis and blotted onto polyvinylidene difluoride membranes. The membranes were overlayed with sonicated leaf midrib extracts from ‘ Ca. L. asiaticus’-infected citrus plants. The protein complexes were detected using different antibodies against ‘ Ca. L. asiaticus’ membrane proteins and peptidoglycans associated with lipoproteins. By comparison, spots were extracted from a polyacrylamide gel for liquid chromatography-tandem mass spectrometry analysis. Identified proteins included ATP synthase alpha and beta subunits, actin, alpha- and beta-tubulins, transitional endoplasmic reticulum ATPase TER94, 78 kDa glucose-regulated protein, and arginine kinase. These findings emphasize the involvement of energy machinery and muscle proteins in the ‘ Ca. L. asiaticus’– D. citri interactions. Understanding the pathogen–vector interactions will lead to better-designed control strategies based on the interference with specific interactions and/or blocking transmission.

Abstract Phytoplasmas are wall-less plant pathogens characterized by highly reduced genomes and limited metabolic capabilities, leading to an obligate host dependency. Since phytoplasma axenic cultivation has not yet been achieved, genomic approaches are essential to unravelling their biological and pathogenic traits. However, such insights are currently hindered by the scarcity of complete genomes and the lack of data for several clades, as is the case for the quarantine-relevant ‘ Candidatus Phytoplasma phoenicium’ (group 16SrIX-B), the causal agent of the severe stone-fruit disease almond witches’ broom (AlmWB). In this study, we present the complete genome sequence of the Lebanese strain F1A, which consists of a 552,248 bp chromosome with a 24.29% GC content and encodes 450 protein-coding genes. Comparative analyses with 16SrIX group draft genomes revealed high conservation among Lebanese AlmWB strains; however, comparisons with related 16SrIX-C phytoplasmas were limited by their low assembly completeness. Beyond providing a complete reference genome for this phytoplasma group, the F1A chromosome revealed distinct metabolic features. Strain F1A lacks the upper glycolytic pathway, indicating a metabolism that relies on glycerol-3-phosphate uptake and carboxylic acid fermentation. Notably, in addition to the conserved malate-to-acetate pathway, F1A encodes a complete citrate lyase complex, suggesting the potential for citrate utilization. Phylogenetic analysis of the associated 2-hydrocarboxylate symporter revealed the widespread occurrence of two phylogenetically distinct variants in phytoplasmas. Apart from the deduced metabolic capacities, the predicted effector repertoire shows similarities with those of other phytoplasmas, including proteins associated with branch proliferation and witches’ broom symptoms. Overall, these findings link genome reduction to host-dependent carboxylic acid metabolism in a quarantine-relevant phytoplasma, establishing a framework for comparative and functional studies of 16SrIX phytoplasmas and providing a basis for future investigations of AlmWB ecology and pathogenicity.

Periploca aphylla witches’ broom (PapWB) is a phytoplasma-associated disease first reported in south-western Iran in 2010. Recent observations showed that the disease is spreading to new areas. PapWB phytoplasmas, detected in symptomatic P. aphylla plants, exhibited identical nucleotide sequences in the 1.8 kb PCR amplicon encompassing the 16S rRNA gene, the 16S-23S spacer region and the 5′-end of the 23S rRNA gene. When compared with the reference strains of previously described ‘ Candidatus Phytoplasma’ species, the selected representative strain PapWB-S exhibited the highest 16S rRNA gene sequence identity (98.15%) with salt cedar witches’ broom (SCWB1) phytoplasma, the reference strain of ‘ Ca . P. tamaricis’. Moreover, the sequence identity values for the tufB, rplV-rpsC and secY genes of PapWB-S strain, when compared with those of reference strains of previously described ‘ Ca . Phytoplasma’ species, were below the thresholds adopted for describing new species. Phylogenetic analyses confirmed the close relationship of the strain PapWB-S with SCWB1 followed by other ‘ Ca . Phytoplasma’ species belonging to the ribosomal groups 16SrX, 16SrXX and 16SrXXXIII. Following the revised guidelines for describing ‘ Ca . Phytoplasma’ species, ‘ Ca . P. periplocae’ sp. nov., associated with PapWB disease, is proposed, and the 16S rRNA gene sequence (accessible at GenBank/NCBI accession number OQ819043) is its nomenclatural type.

A Gram-stain-negative, aerobic, capsule-forming strain, designated DAS-389 T , was isolated from soil collected in the Kogelberg Nature Reserve, Western Cape, South Africa. The strain formed small pink colonies, with cells that multiplied by binary division. Optimum growth was observed at 20 °C to 26 °C and pH 5.5 to 6.5, and it tolerated a NaCl concentration up to 1.5% (w/v). DAS-389 T is chemoorganotrophic, with growth substrate preferences for sugars and polysaccharides, including fructose and starch, respectively. The genome of DAS-389 T had a genome size of 4,136,282 bp with an average G+C content of 63.49 mol% and encodes for an assortment of enzymes, carbohydrate metabolic pathways and several genes involved in stress and survival responses. Major fatty acids (>5%) of strain DAS-389 T were iso-C15:0, iso-C17:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and summed feature 9 (iso-C17:1 ω9c and/or C16:0 10-methyl). Comparative analysis of 16S rRNA gene sequences revealed that strain DAS-389 T shared the highest similarity with Alloacidobacterium dinghuense 4Y35 T (96.6%), followed by Acidipila rosea AP8 T (96.6%) and Paracidobacterium acidisoli 4G-K13T (96.3%). Phylogenetic analysis based on 16S rRNA gene sequences and the whole proteome placed DAS-389 T within a distinct clade, separate from recognized genera in the family Acidobacteriaceae . We therefore propose that strain DAS-389ᵀ represents a novel genus and species, Candidatus Dedyshia acidiphila gen. nov., sp. nov., within the family Acidobacteriaceae , order Terriglobia . The whole-genome sequence of strain DAS-389ᵀ [accession number: JBMGJV000000000 (PRJNA1221820) ] is designated as the nomenclatural type.

A phytoplasma associated with poinsettia has previously been reported in Korea, but the report primarily focused on stem flattening symptoms and was based solely on partial 16S ribosomal RNA (rRNA) gene sequence analysis. In this study, phytoplasma-infected poinsettias collected in Jinan and Jeonju, Korea were examined for diverse symptom expression and additional molecular characterization of the associated phytoplasma. All seven plants showed a free-branching phenotype. Symptom severity ranged from low freebranching, with larger leaves and long internodes, to severe free-branching, with shortened internodes and small, irregularly twisted leaves. Based on 16S rRNA gene sequence and iPhyClassifier analyses, the phytoplasma detected in this study was assigned to subgroup 16SrIII-A of the X-disease phytoplasma group (16SrIII). Although this assignment differs from the previous Korean report, which classified poinsettia-associated phytoplasmas subgroup 16SrIII-H, the present study interpreted the classification using an updated 16SrIII subgroup framework. Sequence analyses of more variable regions, including the 16S-23S intergenic spacer region, ribosomal protein (rp) operon, and secA gene, showed 99.46-99.55% nucleotide identity with ‘Candidatus Phytoplasma pruni’-related reference strains. Phylogenetic analyses showed that the poinsettia-associated phytoplasmas in Korea were closely related to the subgroup 16SrIII-A. These results expand the known symptom spectrum of phytoplasma-infected poinsettias and provide multilocus molecular characterization supporting the phylogenetic placement of poinsettia-associated phytoplasmas in Korea.

Abstract “ Candidatus Liberibacter solanacearum” (Lso) is a phloem-limited bacterial pathogen transmitted by psyllid. In the USA, the haplotypes A and B are transmitted by the tomato psyllid, Bactericera cockerelli, and are responsible for economic damage to the solanaceous crop industry. In tomato, haplotypes A and B cause diseases with differing degrees of severity, yet their underlying mechanisms are not well understood. The role that abiotic stress plays in disease development has not been characterized. In the present study, we evaluated whether water stress influenced the development of symptoms associated with these haplotypes in tomato ( Solanum lycopersicum “Moneymaker”). First, pot-grown tomato plants were subjected to either a control or a water-stress regime, then they were assigned to one of three infection treatments (Lso-free, LsoA, or LsoB) under laboratory-controlled conditions. Symptom development was monitored for up to 8 weeks following infection. Results of this experiment revealed that water stress significantly reduced the plant water potential and was the primary driver of reduced height, with infection only having an effect in later weeks. Water stress also increased the detectability of Lso throughout the plant and across three timepoints (weeks 4, 6, and 8 after infection). This increase in Lso-positive tissues suggests that water stress creates a more homogenous distribution of Lso throughout the plant. More noticeably, water stress significantly reduced the severity of the disease associated with haplotype A but not B. Overall, these findings revealed the existence of haplotype-specific interactions between Lso infection and water stress on the tomato health. This work highlights the influence of abiotic factors on plant health.

Ticks are important vectors of bacteria belonging to the order Rickettsiales, several of which are causes zoonotic diseases. Amblyomma triste is the main vector of Rickettsia parkeri , the etiological agent responsible for most cases of human spotted fever rickettsiosis in Argentina. However, information on the circulation of rickettsial agents in protected areas of the La Plata River Hydrographic Basin, a region that includes highly urbanized environments and frequent contact between wildlife, domestic animals and humans, remains limited. The aim of this study was to investigate the presence of rickettsial bacteria in A. triste collected in protected areas of Buenos Aires Province, (La Plata River Hydrographic Basin, Argentina). Between September and December 2021, ticks were collected by vegetation flagging in four protected areas and analyzed individually. Polymerase chain reactions (PCR) for detecting the genus Rickettsia was initially performed using a simple PCR to amplify a variablesized fragment of the 23S-5S rRNA intergenic spacer. An initial PCR was performed with primers for a 16S rRNA fragment for the Anaplasmataceae family. A total of 247 adult ticks were examined. Twelve specimens (4.9%) tested positive for Rickettsia , and sequence analysis confirmed their identity as R. parkeri . Positive ticks were detected in Ciervo de los Pantanos National Park and Campos del Tuyú National Park. In addition, two specimens yielded sequences corresponding to Candidatus Midichloria sp. These findings confirm the presence of R. parkeri in A. triste populations from protected areas of the La Plata River Hydrographic Basin and provide the first evidence of Ca. Midichloria sp. in this tick species in Argentina. The results highlight the need for continued surveillance of tick-borne microorganisms in protected areas located near densely populated regions, where human exposure to infected ticks may occur.