Publications

4366

Background New tick-borne pathogens are being discovered worldwide, and recognized tick-borne diseases are becoming increasingly diverse. Candidatus R. jingxinensis is endemic in Asia, but its potential to cause clinical infection in humans remains unclear. This study was designed to elucidate the prevalence and delineate the clinical profile of Candidatus Rickettsia jingxinensis infection in Liaoning Province, China. Methods The subjects of this study were suspected cases of tick-borne infectious diseases admitted to the First Affiliated Hospital of China Medical University or reported to the Liaoning Provincial Center for Disease Control and Prevention in 2018–2022. Epidemiological and clinical data were collected. Tick-borne pathogens were detected with a microfluidic chip detection system, and specific gene fragments of the screened pathogens were amplified, sequenced, and compared. Evolutionary and phylogenetic trees were constructed and analyzed. Results In total, 398 infected subjects from 14 cities were included in the study, and 255 tick-borne pathogens were detected. Among these, 11 subjects were found to be infected with Candidatus Rickettsia jingxinensis. This is the first time this strain has been shown to cause infection and illness in humans. The main clinical features of subjects infected with Candidatus R. jingxinensis included fever, fatigue, dizziness, headache, nausea, diarrhea, general pain or muscle and joint pain, reduced leukocytes and platelets, abnormal coagulation function and liver function. Conclusions This study documents the first human infections with Candidatus R. jingxinensis, confirms its prevalence in Liaoning Province, and delineates the primary clinical manifestations of the disease.

Abstract Cable bacteria are filamentous microorganisms capable of centimeter‐scale electron transport, which have great impacts on sediment biogeochemistry, especially oxygen consumption and sulfide depletion. While 16S rRNA sequences related to known cable bacteria have been identified in saline lakes, their genomic diversity, metabolic potentials, and evolution remain unknown. Eight cable bacteria genomes were retrieved from 23 sediment metagenomes across four saline lakes, representing five novel species adapted to different salinity niches. A deep‐branching Electronema species, named Electronema qinghaiense , was found preferentially in brackish to saline environments, implying an ecological and evolutionary link between marine and freshwater lineages. Based on genome analysis, the three newly named cable bacteria species are likely mixotrophic diazotrophs capable of degrading diverse complex carbohydrates, while also participating in hydrogen metabolism via various groups 3 and 4 [NiFe]‐hydrogenases. Genome streamlining and horizontal gene transfer likely drove ecophysiological differentiation among these Electrothrix and Electronema species, including an interphylum horizontal transfer of glycine/sarcosine N‐methyltransferase ( gsmt ) and sarcosine/dimethylglycine N‐methyltransferase ( sdmt ) genes into their common ancestor. Subsequent loss of these genes in some descendants led to adaptation to different salinity niches. Given the inferred ancestral physiological properties, phylogenomic analysis and the evidence that “freshwater” Electronema species experienced stronger purification selection than “saline” Electronema and “hypersaline” Electrothrix species, the evolutionary progression of cable bacteria occurred most likely in the saline‐to‐freshwater direction. Additionally, cable bacteria ecotypes adapted to specific salinity niches likely formed from selective sweeps with low homologous recombination. Collectively, these findings deepen our understanding of the ecophysiology and evolution of cable bacteria.

Cultivation of prickly pear (Opuntia ficus-indica L., family Cactaceae) is of high value in dry-land agriculture in Jordan. In May 2021, symptoms including thickening and severe stunting of the cladodes and deformation of fruits were observed on prickly pear plants cultivated in southern Jordan, Madaba region (31.593565 N, 35.850111 E), with a 15% incidence across three cactus fields. To verify the occurrence of a graft-transmissible disease, wedge grafting was performed on asymptomatic opuntia rootstocks, resulting in thickened cladodes and deformed fruits within five weeks. Samples of cladodes from naturally infected plants were collected from fourteen symptomatic and one asymptomatic plant. A nested polymerase chain reaction (PCR) performed by using primer pair P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995), followed by R16F2n/R16R2 (Gundersen and Lee 1996), amplified a fragment of the expected size only from the symptomatic samples. Direct amplicon sequencing followed by blast comparison to both, GenBank and EPPO-QBank databases (https://qbank.eppo.int/phytoplasmas/) allowed the identification of a ’Candidatus Phytoplasma australasiae=australasiaticum’ strain (GenBank accession no. PQ319761, designated strain ´Cact1´), with a 100% sequence identity to the reference strain (GenBank accession no. Y10097, ribosomal subgroup 16SrII-D) (White et al., 1998; Rodrigues et al., 2023). The phylogenetic analysis of strain ´Cact1´ with phytoplasma strains of ribosomal subgroups in group 16SrII and detected in cactus in China, Italy and Turkey showed that strain ´Cact1´ is not clustering with any of them (Figure 1). Indeed, comparison with all sequences in GenBank, show that the phytoplasma from Jordan clusters with those enclosed in the 16SrII group under the species ‘Ca. P. australasiae=australasiaticum’, showing about 99.00% identity to this phytoplasma and having an identity to ‘Ca. P. aurantifolia=citri’ of about 98.50%, that is below the accepted threshold for ‘Ca. Phytoplasma’ strains differentiation (Bertaccini et al. 2022). This identification was confirmed by amplifying and sequencing the leucyl transfer RNA synthetase (leuS) gene (Abeysinghe et al. 2016) (GenBank accession no. PQ349195), that showed 100% sequence identity with 100% coverage to the Parthenium hysterophorus phyllody phytoplasma strain ´PR08´ from India (GenBank accession no. CP097207), identified as a strain of ‘Ca. P. australasiae=australasiaticum’ (White et al., 1998; Rodrigues et al., 2023). Other phytoplasma strains having 100% identity on the leuS gene but with 95-96% coverage were reported in pearl millet, soybean and alfalfa from India (GenBank accession no. MW020555, MW020562 and MW020559 respectively). The leuS gene sequence has been relevant to confirm the identification of phytoplasmas infecting various agricultural important crops especially in Asian countries (Tiwari et al., 2023). It is thus necessary to investigate insect vector(s) presence and search for other economically important hosts and for alternative host weeds for this phytoplasma considering that the disease and the associated phytoplasmas are present and spreading in other regions of Jordan.

Big Basin Sagebrush (Artemisia tridentata ssp. tridentata) is a common shrub found across high desert and arid regions of North America. Although its natural distribution has been reduced over the past century due to farm and urban expansion it remains a keystone species in much of the high desert and is a host for native arthropod species. However, there have been few studies that examined the effect of phytopathogens on A. tridentata (Allen & West 1987; Welch & Nelson 1995). During a survey for alternate hosts of the X-disease phytoplasma (‘Candidatus Phytoplasma pruni’) (Shires et al. 2025) we observed phytoplasma-like foliar chlorosis and dieback-like symptoms on A. tridentata in uncultivated areas of Chelan County, WA. Approximately 30-40% of plants were symptomatic at these sites, and symptom progression was seasonal, with the emergence of enlarged, pale-yellow chlorotic leaves between late spring (May-June) and summer (July-August). Chlorosis began at the leaf apex and extended towards the petiole, with tip necrosis then leaf drop occurring in late August (Figure 1). Dieback of shoot apices and limbs was also observed on individual plants. Leaf and stem tissues were collected from 19 symptomatic and 63 asymptomatic sagebrush at three sites in Chelan Co., DNA extracted using the Qiagen DNeasy plant mini kit (Qiagen Corp. Germantown, MD) per the manufacturer’s instructions, and tested for 16SrIII phytoplasmas on the basis of proximity to orchards known to be infected with ‘Ca. P. pruni’ by qPCR as per Shires et al. (2025). All symptomatic plants tested positive, while asymptomatic plants did not. For identification, the partial 16S rRNA gene was amplified from seven samples by nested PCR using the P1/P7 (Deng & Hiruki 1991) then R16F2n/R2 primers (Gundersen & Lee 1996); resulting products were Sanger-sequenced (Eton Biosciences, San Diego, CA) and deposited in GenBank (PV994682-PV994688). Examination using iPhyClassifier (Zhao et al. 2012) indicated that these were a ‘Ca. P. pruni’-related strain with 99.66% identity to reference sequence JQ0443393. BLASTn indicated a 100% nucleotide identity match to Lilac decline phytoplasma strain LlcDec1 (KP877578) (Davis et al. 1996). For further identification, the partial secY (849 bp), secA (490 bp), Ef-Tu (793 bp), and imp (457 bp) genes were amplified and sequenced as per Molnar et al. (2024). Of these secY (PX023655-PX023661), secA (PX023662-PX023668) and Ef-Tu (PX023669-PX023675) had 99.8%, 98.9% and 98.5% identity to X-disease strains PX11CT1 (JQ268254), 20-15 (OR67122), and 22-2470 (OR670177) respectively, whereas imp (PX023676-PX023682) had 96.9 % identity to PoIBI strain “Annette Hegg Maxi” (AB636364) (100% coverage for all fragments). Neighbor-joining phylogenetic analysis of the concatenated marker regions showed that this phytoplasma forms a separate clade from other 16SrIII strains, and whose closest relative is PoiBI strain JR1 (ASM30946v1) with 98.9% identity across the aligned region (Figure 2). Cumulatively these data suggest the Sagebrush yellows-associated phytoplasma is a ‘Ca. P. pruni’-related strain that is novel and distinct from previously described members of the 16SrIII group, based on sequence identity and the host on which it causes disease. This find is significant as it is the first report of a novel phytoplasma infecting a keystone species in western North America and an indicator of where 16SrIII phytoplasmas infecting cultivated plants may have originated.

ABSTRACT Phytoplasmas were detected in potato tubers and sugar beet roots in neighbouring fields in Southern Germany using quantitative PCR. Infected potato plants showed phytoplasma‐associated symptoms including yellowing, upward leaf rolling, aerial tubers and early senescence. Sugar beet plants showed similar symptoms to those of syndrome ‘basses richesses’ (SBR) disease, including proliferation, yellowing and necrosis of older leaves. The genetic diversity of the phytoplasmas in the symptomatic potato and sugar beet plants was investigated through sequence analysis of 16S rRNA and stamp , vmp1 , tuf and secY genes. The RFLP profiles and sequences of 16S rRNA were identical among all the sugar beet‐positive samples and aligned with subgroup 16SrXII‐P, whereas the potato phytoplasma strains were identified as belonging to subgroup 16SrXII‐A. Sequence analysis of the non‐ribosomal genes showed that the potato strains belonged to the b1 group of the tuf gene, the V4 and V17 groups of the vmp1 gene and the secY3 and secY4 groups of the secY gene. However, the sugar beet strain was clearly distinct from the potato strains. Together with strain 916/22 from sugar beet in eastern Germany, they formed a new group of tuf , secY , vmp and stamp genes. RFLP assays and multilocus sequence analysis of non‐ribosomal genes of the phytoplasma strains confirmed that the potato phytoplasma strains were molecularly different from the sugar beet phytoplasma strains. In addition, this is the first report on the multilocus sequence analysis of ‘ Candidatus Phytoplasma solani’ strains in potato plants in Germany, suggesting differences in source plants other than sugar beet or interactions with insect vectors specific to each crop.

ABSTRACTDespite recent genomic studies and increased molecular data, epitheliocystis remains an enigmatic fish disease with no experimental in vitro or in vivo models to aid the advancement of research. In this study, we revert to a classical microscopical approach and screen with the electron microscope the epitheliocystis lesions caused by a Ca. Parilichlamydia sp., infecting mucus cells in Greater amberjack. We report distinct morphological features of this bacterial family, characterised by Intermediate Bodies that closely resemble those of previously described Candidatus similchlamydia, and Elementary Bodies that exhibit morphological similarities to Chlamydia trachomatis. We describe the characteristics of a novel Chlamydial Inclusion Membrane (IM) type, with abundant interdigitations, possibly shaped by fusion of the IM with cytoplasmic vesicles, and moreover discuss the presence of multivesicular bodies in the infected cell. Our observation of immune cells in the infected areas indicates an interaction of macrophages with infected cells, a role for granular cells as pathogens reservoirs and an active phagoptosis process in the nearby areas, overall shedding light on cellular immune processes characterising these infections in fish hosts.

A Microviridae phage, CLasMV1, was recently identified in ‘ Candidatus Liberibacter asiaticus’ (CLas), the bacterium associated with citrus Huanglongbing (HLB). The CLasMV1 strain GDHZ11D (CP045566) exhibited a circular genome of 8,869 bp containing eight open reading frames (ORFs). Phages of CLas play crucial roles in regulating bacterial population diversity that have potential use for HLB control. However, knowledge of CLasMV1 population diversity is currently limited. In this study, 1,520 CLas samples were collected from 10 citrus-growing provinces in China. PCR screening revealed that 1,042 samples (69%) harbored CLasMV1. Complete genome sequences of 43 CLasMV1 strains were obtained through Illumina HiSeq sequencing with genome sizes ranging from 8,696 to 8,881 bp. Variations were mostly related to single-nucleotide polymorphisms and sequence insertions/deletions. Notably, two hypervariable regions (HVRs) were identified. Two new ORFs were detected, ORF-A (144 bp) in HVR-1 and ORF-B (342 bp) in HVR-2. Whole-genome phylogenetic analysis using 43 strains from this study, 9 previously reported Chinese strains, and 2 Florida strains (CP040636 and MZ382797) delineated five genomic groups. Chinese strains dominated Groups 1 to 4, and Group 5 contained the Florida strains. Further analyses of HVR-1 across all 1,042 CLasMV1 samples detected a 173-bp sequence with suspected mobile activity. Analysis of a PacBio long-read assembly published previously detected a concatemer of the CLasMV1 sequence, suggesting that the phage used the rolling circle mechanism for its replication. These findings significantly enhance our understanding of CLasMV1 genomic diversity and will facilitate future research on CLasMV1 classification and HLB biology.