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Citrus greening disease, also known as huanglongbing (HLB), is caused by the gram-negative α-proteobacteria Candidatus Liberibacter species. This disease poses a significant threat to citrus production worldwide, including in Nepal. This study aimed to perform the diagnosis and phylogenetic analysis of the citrus greening pathogen in Nepal using both conventional PCR and computational methods. A total of 1,026 citrus leaf samples were collected from thirteen districts across six provinces in the country. PCR-based diagnosis was performed using the primer set Las606/LSS, which targets the 16S rRNA gene of Candidatus Liberibacter asiaticus. The reliability of the PCR was validated by including previously confirmed positive and negative controls in every run. Furthermore, each assay was performed in triplicate to ensure consistency and reproducibility of results. Additionally, 16S rRNA gene sequencing was performed using Sanger sequencing for five samples. The obtained sequences were deposited in GenBank and a phylogenetic tree was constructed based on these sequences. Among the 1,026 samples tested, 255 were positive, indicating the widespread distribution of HLB across Nepal. All consensus sequences from Nepal showed strong evolutionary relatedness within the Ca. L. asiaticus cluster, with over 99% genetic similarity to reference sequences from various parts of the world. Phylogenetic analysis revealed that the Nepalese sequences were closely related to Ca . L. asiaticus sequences from India (Punjab and Meerut) and sequences obtained from different regions of Nepal clustered closely together. The molecular findings from this study reveal a high prevalence of citrus greening disease across Nepal and underscore the urgent need for integrated management policies, including the use of certified clean planting material and vector (psyllid) control programs. The generated sequence data serves as a vital resource for developing regional diagnostic tools and guiding future surveillance strategies to mitigate HLB’s impact on the world’s citrus industry.

Citrus Huanglongbing (HLB), a devastating disease caused by the Gram-negative bacterium ‘ Candidatus Liberibacter asiaticus’ ( C Las), poses serious threats to global citrus production and lacks effective control strategies. Previously, SDE2470 (CLIBASIA_02470) was identified as a Sec-dependent effector that contributes to C Las pathogenesis, although its underlying molecular mechanisms were not fully elucidated. In this study, SDE2470 was found to target a citrus vascular one-zinc-finger transcription factor CsVOZ2. CsVOZ2 overexpression ( CsVOZ2- OE) in transgenic citrus plants significantly suppressed C Las colonization, whereas its RNA interference (RNAi) in citrus hairy roots enhanced susceptibility to C Las. Additionally, CsVOZ2 -OE significantly increased reactive oxygen species (ROS) and abscisic acid (ABA) contents accumulation and activated related genes expression. Further investigation revealed that the E3 ligase CsBTS1 directly interacts with CsVOZ2 and promotes its degradation via the 26S proteasome pathway. CsBTS1E3 -OE in citrus hairy roots markedly enhanced C Las proliferation. Importantly, SDE2470 directly interacts with CsBTS1E3 and strengthen CsBTS1E3-CsVOZ2 interaction. Meanwhile, SDE2470 strengthened the E3 ligase activity of CsBTS1, promoting CsVOZ2 degradation. Taken together, these findings support a model in which SDE2470 hijacks CsBTS1 to destabilize CsVOZ2, thereby disrupting ROS- and ABA-dependent immunity and promoting C Las infection in citrus.

A Gram-negative, facultative anaerobic, rod-shaped, motile with peritrichous flagella, fluorescent bacterium, designated ‘Candidatus Pseudomonas auctus’ sp. nov. JDE115, was isolated from soybean root nodules in Virginia and characterized using a comprehensive integrative methodology. Growth of JDE115 occurred with 0–5.0% (w/v) NaCl (optimum 1%), at pH 6.0–10.0 (optimum pH 7.0), and at 10–40°C (optimum 28°C) in LB broth. Phylogenetic analyses based on the 16S rRNA gene placed the isolate as a member of a novel species within the genus Pseudomonas. Phylogenetic analyses based on whole-genome sequences, 16S rRNA, showed JDE115 having the highest similarity to Pseudomonas glycinae MS586. Average Nucleotide Identity (ANI) analysis also revealed the highest similarity of JDE115 to Pseudomonas glycinae MS586 (94.59%), which is below the 95% threshold for species delineation. Genome-to-genome distance analysis (GGDC, Formula 2) showed a maximum value of 57.10% with the same strain, far below the 70% cutoff. The primary isoprenoid quinone detected in JDE115 was ubiquinone-9 (Q-9) and the DNA G + C content was 60.68 mol%. The whole-cell fatty acid profile was dominated by C16:0, C17:0 cyclo, and the summed features 3 (C16:1ω7c and/or C16:1ω6c) and 8 (C18:1ω7c and/or C18:1ω6c). Additional fatty acids detected included 12:0, 14:0, and 18:0. Based on these phenotypic, chemotaxonomic, and phylogenetic data, strain JDE115 is proposed to represent a new species in the genus Pseudomonas, for which the name ‘Candidatus Pseudomonas auctus’ sp. nov. is proposed.

Huanglongbing (citrus greening disease) is caused by the bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) (Alphaproteobacteria) and is one of the most destructive bacterial-vector diseases affecting the citrus industry. The bacterium is transmitted by the Asian citrus psyllid (ACP; Diaphorina citri). Early detection in citrus trees is challenging due to uneven distribution of CLas throughout the tree and a long pre-symptomatic phase of the disease. Due to these limitations, ACP sampling has been suggested as a more reliable early detection strategy. The objective of this study was to develop and optimize approaches for detecting CLas in ACP adults and nymphs collected in citrus groves in California using real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). The goal was to establish the optimal number of ACP adults and nymphal instar life stages (stages 1–2, 3, or 4–5) that yielded the most reliable detection of CLas (Cq values ≤ 38). Results indicated that CLas detection correlated with psyllid developmental stage, with the 4th–5th instar nymphs (sample size of five to ten per tube) or adult ACP (sample size of three to ten per tube) providing the most consistent qPCR detection. While CLas detection rates increased with adult ACP age, nymphs were preferred for field sampling as adult ACP might have dispersed from non-infected trees, potentially misrepresenting the grove’s CLas status. Detection by droplet digital PCR confirmed the presence and genome copies of CLas in a subset of ACP across life stages. In field populations, detection rates in nymphs were consistent or stable throughout the year, whereas CLas detection in adults exhibited seasonal variation, with CLas detection and genome load peaking in January. These targeted ACP sampling strategies and optimized laboratory processing methods will facilitate CLas detection in psyllids for streamlining citrus greening disease management.

Background Candidate Phyla Radiation (CPR) is a large monophyletic group encompassing about 25% of bacterial diversity. Among CPR, “Candidatus Saccharibacteria” is one of the most clinically relevant phyla. Indeed, it is enriched in the oral microbiota of subjects suffering from immune-mediated disorders and it has been found to have immunomodulatory activities. For these reasons, it is crucial to have reliable methods to detect and quantify this bacterial lineage in human samples, including saliva. Methods and results Four qPCR protocols for quantifying “Ca. Saccharibacteria” (one targeting the 23S rRNA gene and three the 16S) were tested and compared. The efficiency and coverage of these four protocols were evaluated in silico on large genomic datasets, and in vitro on salivary DNA samples, already characterized by amplicon sequencing on the V3-V4 regions of the 16S rRNA. In silico PCR analyses showed that all qPCR primers lose part of the “Ca. Saccharibacteria” genetic variability, even if the 23S qPCR primers matched more lineages than the 16S qPCR primers. In vitro qPCR experiments confirmed that all 16S-based protocols strongly underestimated “Ca. Saccharibacteria” in salivary DNA, while the 23S qPCR protocol gave quantifications more comparable to 16S amplicon sequencing. Conclusion Overall, our results show that the 23S-based qPCR protocol is more precise than the 16S-based ones in quantifying “Ca. Saccharibacteria”, although all protocols probably underestimate specific lineages. These results underline the current limits in quantifying “Ca. Saccharibacteria”, highlighting the needs for novel experimental strategies or methods. Indeed, the underestimation of “Ca. Saccharibacteria” in clinical samples could hide its role in human health and in the development of immune-mediated diseases.

Our previous studies indicate the abundant and diverse presence of yet-to-be-cultured microorganisms in the micropore-filtered fractions of various environmental samples. Here, we isolated a novel bacterium (designated as strain TMPK1T) from a 0.45-μm-filtered soil suspension by using a gel-filled microwell array device comprising 900 microwells and characterized its phylogenetic and physiological features. This strain showed low 16S rRNA gene sequence identities (<91%) and low average nucleotide identity values (<70%) to the closest validly described species, and belonged to a novel-family-level lineage within the orderRhodospirillalesofAlphaproteobacteria. Strain TMPK1Texhibited small cell sizes (0.08–0.23 μm3) and had a high cyclopropane fatty acid content (>13%), and these characteristics were differentiated from otherRhodospirillalesbacteria. A comprehensive habitability search using amplicon datasets suggested that TMPK1Tand its close relatives are mainly distributed in soil and plant-associated environments. Based on these results, we propose that strain TMPK1Trepresents a novel genus and species namedRoseiterribacter gracilisgen. nov., sp. nov. (JCM 34627T= KCTC 82790T). We also proposeRoseiterribacteraceaefam. nov. to accommodate the genusRoseiterribacter.

Ciliates are unicellular eukaryotes, regularly involved in symbiotic associations. Symbionts may colonize the inside of their cells as well as their surface as ectosymbionts. Here, we report on a new ciliate species, designated asZoothamnium mariellasp. nov. (Peritrichia, Sessilida), discovered in the northern Adriatic Sea (Mediterranean Sea) in 2021. We found this ciliate species to be monospecifically associated with a new genus of ectosymbiotic bacteria, here proposed asCandidatusFusimicrobium zoothamnicola gen. nov., sp. nov. To formally describe the new ciliate species, we investigated its morphology and sequenced its 18S rRNA gene. To demonstrate its association with a single species of bacterial ectosymbiont, we performed 16S rRNA gene sequencing, fluorescencein situhybridization, and scanning electron microscopy. Additionally, we explored the two partners’ cultivation requirements and ecology.Z. mariellasp. nov. was characterized by a colony length of up to 1 mm. A consistent number of either seven or eight long branches alternated on the stalk in close distance to each other. The colony developed three different types of zooids: microzooids (“trophic stage”), macrozooids (“telotroch stage”), and terminal zooids (“dividing stage”). Viewed from inside the cell, the microzooids’ oral ciliature ran in 1 ¼ turns in a clockwise direction around the peristomial disc before entering the infundibulum, where it performed another ¾ turn. Phylogenetic analyses assignedZ. mariellasp. nov. to clade II of the family Zoothamnidae. The ectosymbiont formed a monophyletic clade within theGammaproteobacteriaalong with two other ectosymbionts of peritrichous ciliates and a free-living vent bacterium. It colonized the entire surface of its ciliate host, except for the most basal stalk of large colonies, and exhibited a single, spindle-shaped morphotype. Furthermore, the two partners together appear to be generalists of temperate, oxic, marine shallow-water environments and were collectively cultivable in steady flow-through systems.

Ticks are important vectors of zoonotic diseases and play a major role in the circulation and transmission of many rickettsial species. The aim of this study was to investigate the carriage of Candidatus Rickettsia tarasevichiae (CRT) in a total of 1168 ticks collected in Inner Mongolia to elucidate the potential public health risk of this pathogen, provide a basis for infectious disease prevention, control and prediction and contribute diagnostic ideas for clinical diseases that present with fever in populations exposed to ticks. A total of four tick species, Haemaphysalis concinna (n = 21), Dermacentor nuttalli (n = 122), Hyalomma marginatum (n = 148), and Ixodes persulcatus (n = 877), were collected at nine sampling sites in Inner Mongolia, China, and identified by morphological and molecular biological methods. Reverse transcription PCR targeting the 16S ribosomal RNA (rrs), gltA, groEL, ompB and Sca4 genes was used to detect CRT DNA. Sequencing was used for pathogen species confirmation. The molecular epidemiological analysis showed that three species of ticks were infected with CRT, and the overall positive rate was as high as 42%. The positive rate of I. persulcatus collected in Hinggan League city was up to 96%, and that of I. persulcatus collected in Hulun Buir city was 50%. The pool positive rates of D. nuttalli and H. marginatum collected in Bayan Nur city and H. concinna collected in Hulun Buir city were 0%, 28% and 40%, respectively. This study revealed the high prevalence of CRT infection in ticks from Inner Mongolia and the first confirmation of CRT detected in H. marginatum in China. The wide host range and high infection rate in Inner Mongolia may dramatically increase the exposure of CRT to humans and other vertebrates. The role of H. marginatum in the transmission of rickettsiosis and its potential risk to public health should be further considered.

Symbiotic relationships are ubiquitous throughout the world’s oceans, yet for many marine organisms, including those in the high latitudes, little is understood about symbiotic associations and functional relationships. From a recently determined genome sequence of a filter-feeding basket star from Argentina, Gorgonocephalus chilensis, we discovered a novel Mycoplasma species with a 796Kb genome (CheckM completeness of 97.9%, G+C content = 30.1%). Similar to other Mycoplasma spp. within Mycoplasmatota, genomic analysis of the novel organism revealed reduced metabolic pathways including incomplete biosynthetic pathways, suggesting an obligate association with their basket star host. Results of 16S rRNA and multi-locus phylogenetic analyses revealed that this organism belonged to a recently characterized non-free-living lineage of Mycoplasma spp. specifically associated with marine invertebrate animals. Thus, the name “Candidatus Mycoplasma mahonii” is proposed for this novel species. Based on 16S rRNA PCR-screening, we found that Ca. M. mahonii also occurs in Gorgonocephalus eucnemis from the Northwest Pacific and other Gorgonocephalus chilensis from Argentinian waters. The level of sequence conservation within Ca. M. mahonii is considerable between widely disparate high-latitude Gorgonocephalus species, suggesting that oceanic dispersal of this microbe may be greater than excepted.

Citrus Huanglongbing (HLB) is one of the most destructive diseases of citrus plants caused by the obligate and phloem-limiting bacterium Candidatus Liberibacter asiaticus (Las). Reliable detection methods are important for successful control of the disease. This study was aimed to develop a rapid and user-friendly on-site detection system for Las using the TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) assay. The Las-specific on-site detection system could be completed within one hour by simple DNA extraction coupled with a portable POCKIT device, which can perform PCR amplification and automatically provide qualitative results derived from fluorescence signals. The sensitivity of the TaqMan probe-iiPCR assay could be as low as single copy of Las, comparable to a real-time PCR method. Further testing of the field citrus samples showed 100% agreement between the TaqMan probe-iiPCR assay and the real-time PCR method, and the on-site detection system also demonstrated a great performance of Las detection. With high specificity and sensitivity, the on-site detection system developed in this study becomes a simple, rapid and powerful tool for detecting Las in fields.