Publications

4368

Sweet potato witches' broom disease caused by phytoplasmas threatens crop productivity in Fujian, China. Here we report a complete phytoplasma genome consisting of a single circular chromosome (680,179 bp with a G+C content of 29.44%) via the next-generation sequencing of sweet potato witches’ broom-infected samples. A total of 730 genes were predicted, while two 16S rRNAs were revealed to be identical to sequences in ‘Candidatus Phytoplasma australasiaticum’. Following the latest guidelines for the description of phytoplasma species, we have named the strain Candidatus Phytoplasma australasiaticum SPWBPT2024. The genome contained 33 transporters, 35 putative effectors (including SAP11 and SAP54 with multiple copies), and two potential mobile units. Analyses of published peanut and sweet potato phytoplasma genomes revealed the similarities between their effectors and the effectors in this phytoplasma genome. A motif analysis was performed to clarify the difference between the two SAP54 copies in the obtained phytoplasma genome as well as the trends in conserved motifs in other strains. In addition, metabolic pathway-related genes in three genomes were examined, revealing deficiencies in the glycolysis and pyrimidine synthesis pathways. A pan-genome analysis has revealed that the phytoplasma genome is an open genome. To gain further insights into whole-cell metabolic processes, we reconstructed draft genome-scale metabolic models (GEMs) of the genome. This study provides valuable genomic insights into this phytoplasma, characterising conserved and strain-specific SAP effectors, as well as distinct metabolic deficiencies that may help advance research to combat phytoplasma infections, including accurate diagnostics, vector transmission control and antimicrobial interventions.

Insecticide applications are commonly recommended for managing Diaphorina citri, the vector of huanglongbing (HLB), but their effectiveness in reducing transmission of ‘Candidatus Liberibacter asiaticus’ (CLas), especially during continuous psyllid influx and shoot growth, remains unclear. This study evaluated the efficacy of foliar application of thiamethoxam and spinetoram in reducing CLas transmission in sweet orange seedlings. Two experiments were conducted up to 13th days after first spray. In experiment #1, CLas-positive psyllids were confined in seedlings every two days. Treatments were: control; S14 — one spray at day 0; and S7 — sprays at days 0 and 7. In experiment #2, psyllids were released for free choice within a screenhouse every two days. Treatments were: control; S14; S7; and S3 — sprays at day 0 and every 3 days. Sprays on day 0 were over unfolded leaves (V2 stage). Psyllid mortality was assessed in experiment #1. Psyllid occupancy (percentage of seedlings with at least one psyllid) and abundance (number of psyllids per seedling) were assessed in experiment #2. After 6 months, seedlings were tested for CLas by qPCR. In experiment #1, cumulative psyllid mortality was higher in sprayed treatments than control for both insecticides, but HLB incidence was similar. In experiment #2, both psyllid occupancy and abundance decreased with shorter spray intervals, with the lowest HLB incidence in S3. These findings suggest that reducing spray intervals can limit CLas transmission during periods of shoot growth and continuous vector pressure. However, CLas transmission was not entirely prevented, highlighting the need for integrated, area-wide management strategies.

Brinjal little leaf (BLL) disease, caused by phytoplasmas, leads to significant yield losses, but its pathogenic mechanisms remain poorly understood. This study characterizes SAP54LP/S54LP (SAP54-like protein of BLL), an effector protein associated with the 16SrVI-D phytoplasma strain linked to BLL in Tamil Nadu, India. Molecular analyses, including nested PCR and virtual RFLP, confirmed the phytoplasma's classification within the 16SrVI group. The S54LP effector gene was amplified from infected brinjal, confirming its presence. Sequence comparisons showed that S54LP was 100 % identical to homologs in the 16SrVI-A group and 98 % identical to those in the 16SrIB group (phyl1 and S54LP), indicating group-specific divergence. Comparative analysis identified 13 single nucleotide polymorphisms (SNPs), 69 % of which led to nonsynonymous substitutions in the mature protein, potentially affecting host interactions. Haplotype network analysis revealed a globally distributed Hap_6 lineage and region-specific haplotypes, suggesting localized evolutionary dynamics. Evolutionary analysis indicated strong purifying selection on the coding sequence (Ka/Ks ≈ 0.3), reflecting the conservation of functional domains essential for pathogenicity. Conversely, positive selection was detected in the signal peptide region, implying adaptive evolution related to secretion and host targeting. Additionally, AT-rich codon usage at specific sites highlights multiple layers of evolutionary constraints shaping S54LP. Overall, these findings establish S54LP as a model for studying host-phytoplasma coevolution, providing insights into effector biology and informing strategies to manage phytoplasma diseases in crops.

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.

‘ Candidatus Liberibacter solanacearum’ (Lso), haplotype D, is an insect-transmitted, phloem-limited bacterium that induces developmental abnormalities in carrots, including witches’ broom and hairy root symptoms. We hypothesize that these symptoms result from Lso-induced hormonal imbalances. To investigate this, we analyzed the spatial and temporal distribution of Lso in carrot plants and assessed its effects on hormone-related gene expression and phytohormone levels. Our findings revealed that Lso first accumulates in the shoot apical meristem before spreading to root tissues, aligning with phloem flow dynamics. Transcriptomic and biochemical analyses indicated that cytokinin (CK) biosynthesis and response genes were upregulated, whereas gibberellin biosynthesis genes were downregulated. In contrast, no significant changes were observed in auxin biosynthesis or signaling. Hormone quantification further demonstrated increased CK levels in lateral roots and decreased CK levels in the root meristem of infected plants, with no detectable changes in auxin levels. Additionally, salicylic acid and jasmonic acid were significantly elevated following Lso infection, suggesting a persistent plant defense response. To validate the role of CK and auxin in symptom development, we applied synthetic growth regulators to infected and uninfected plants. CK treatment exacerbated witches’ broom symptoms, whereas auxin application mitigated this phenotype but enhanced lateral root formation. These results suggest that Lso manipulates phytohormone homeostasis to induce disease symptoms, offering a potential avenue for symptom mitigation through targeted hormone applications. Our findings provide novel insights into the role of plant hormones in Lso pathogenesis and highlight new strategies for managing carrot yellows disease.