Publications

4567

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.

Sugar beet cultivation in Europe is threatened by two vector-borne diseases: syndrome “basses richesses”, caused by the phloem-limited pathogen ‘Candidatus Arsenophonus phytopathogenicus’, and phytoplasmoses associated with ‘Ca. Phytoplasma solani’ subgroup 16SrXII-A and the related subgroup 16SrXII-P. Infections lead to reduced sugar yield, biomass and growth abnormalities. In Germany, Pentastiridius leporinus represents the main vector. Despite their importance, genetic diversity remains poorly understood. During a two-year survey, barcoded amplicons were generated from infected sugar beet samples from Germany and neighbouring countries using the phytoplasma markers 16S rRNA-ITS-23S rRNA, tuf, and groEL-stamp-nadE, as well as rplO-secY-rpmJ and groEL for ‘Ca. A. phytopathogenicus’. Amplicon pools underwent single-molecule real-time sequencing and amplicon-sequence-variant inference. Additionally, planthopper samples from sugar beet in Germany were analysed and compared to sugar beet data for ‘Ca. A. phytopathogenicus’. No genetic diversity of ‘Ca. A. phytopathogenicus’ was detected, whereas 16SrXII-A and -P showed variation below the subgroup level. 16SrXII-A exhibited higher diversity than 16SrXII-P. In Germany, 16SrXII-A formed a single cluster, while 16SrXII-P comprised two clusters based on 16S rRNA-ITS-23S rRNA. In neighbouring countries, only 16SrXII-A showed diversity, resolving up to four clusters by groEL-stamp-nadE. These results provide a basis for the identification of dominant strains supporting comparative variety evaluation for tolerance.

Citrus huanglongbing (HLB) is recognized as the most destructive bacterial disease affecting citrus. Among the causal agents, Candidatus Liberibacter asiaticus (Las) is the predominant species causing HLB epidemics worldwide. Detecting this phloem limited bacterium at early or low titer stages and assessing its cellular activity remain major challenges for HLB research and management. To address these limitations, we developed a one step reverse transcription quantitative PCR (RT qPCR) assay for highly sensitive Las detection using total nucleic acids and the established Li 16S rRNA/rDNA primer–probe set. This method simultaneously amplifies both Las 16S rRNA and rDNA in a single reaction, increasing sensitivity by up to 500 fold compared with qPCR, which targets only 16S rDNA. The improved sensitivity reduced false negatives, expanded sampling capacity, and enabled detection of low titer infections previously undetectable. In addition, the higher abundance of 16S rRNA generated a consistent Ct gap between RT qPCR and qPCR, which we used to estimate relative Las cell activity in vivo in host insects and plants and to identify the most effective inoculum sources. This comparative detection strategy was further applied to evaluate inoculum potential, monitor changes in relative cell activity during in vitro cultures, and assess antimicrobial treatments targeting Las. The persistent detection of long lasting, low titer Las infections underscores the complex biology of the HLB causal agent and highlights ongoing challenges for effective disease management.

Abstract Mauritius is one of the two countries where two huanglongbing (HLB)-associated bacteria, ‘ Candidatus Liberibacter asiaticus’ (CLas) and ‘ Ca . Liberibacter africanus’ (CLaf), have coexisted since at least the 1990s, offering insights into their interactions, particularly given their divergent thermal tolerances. The objective of the study was to revisit the unique HLB epidemiological situation in Mauritius using modern diagnostic and genotyping techniques in the broader context of global warming. Using multiple-locus variable number tandem repeat analysis (MLVA), multilocus sequence analysis (MLSA), a real-time PCR assay, and a loop-mediated isothermal amplification (LAMP) assay, we detected both species. While CLas was commonly detected from sea level up to 580 m above sea level (masl), CLaf was commonly detected between 300 and 600 masl, where temperatures appear higher than in African regions where CLaf has been reported, suggesting putative adaptation to warmer temperatures of Mauritian CLaf. LAMP data also identified some limitations in inclusivity in Keremane’s system. Based on tandem-repeat (TR) data, a single population of CLas was identified whose level of diversity did not differ statistically from that of Réunion, a geographically proximate island with a similar epidemiological situation. These two populations were only weakly differentiated, consistent with the detection of very few private alleles in Mauritius. Prophage- and miniature inverted-repeat transposable element (MITE)-based genotyping confirmed the high genetic relatedness between samples from these islands. Altogether, our results suggest a putative identical source for CLas and/or epidemiological links between these islands. We discuss the implications of our results for surveillance and management of HLB.

Abstract Members of the bacterial family Endozoicomonadaceae are ubiquitous marine symbionts associated with diverse hosts, including corals, sponges, molluscs, and fishes. Despite their ecological pervasiveness, inhabiting deep-sea vents and sunlit coral reefs, their taxonomy remains inconsistent, largely due to reliance on 16S rRNA gene phylogenies that fail to fully resolve evolutionary relationships. Here, we integrate phylogenomics, comparative genomics, and overall genome relatedness indices across 63 high-quality genomes to re-define genus boundaries within the Endozoicomonadaceae family. Among the tested metrics, the core-proteome average amino acid identity proved the most robust for genus-level resolution, with a proposed identity cutoff of 76%, aligning with phylogenomic structure and maintaining taxonomic robustness across the family. Phylogenomic reconstructions further revealed that the taxon Endozoicomonas is paraphyletic, forming two well-supported, but distinct clades. This prompts a taxonomic revision, dividing Endozoicomonas into Endozoicomonas sensu stricto and Neoendozoicomonas gen. Nov., with Neoendozoicomonas montiporae designated as the type species. Furthermore, the sister clade to Endozoicomonas sensu stricto - comprising the genera Endonucleibacter and Sororendozoicomonas - is characterized by genomic streamlining, including reduced genome size, lower GC content, reduced number of orthogroups, and potential functional divergences consistent with host specialization. Functional annotations highlighted secretion and conjugation systems as key differentiators between genera, emphasizing potential distinct host-interaction strategies. This genome-based framework refines the taxonomy of Endozoicomonadaceae, provides additional criteria for genus delimitation, and strengthens the evolutionary and ecological interpretation of these widespread marine bacterial symbionts.

ABSTRACT Acidophilic ammonia-oxidizing bacteria (AOB) have only recently been discovered. These organisms hold great promise for acidic wastewater treatment; however, their physiology remains poorly understood compared to that of neutrophilic AOB. Here, we investigated the physiology of the acidophilic AOB “Candidatus Nitrosacidococcus tergens ” sp. RJ19 across a broad pH range (2.5–7.0) using a specialized bioreactor system. We monitored nitrogen (N) transformations combined with microbial community composition and transcriptomic profiles, focusing on nitrogen metabolism and proton stress responses. Our results show that above pH 6.0, “ Ca . Na. tergens ” performs complete and stoichiometric conversion of ammonium to nitrite, coinciding with isotopic fractionation effects specific for ammonia oxidation and increased expression of key ammonia oxidation genes. The apparent absence of nirK and cycA did not impede ammonia oxidation, suggesting that these genes are non-essential in this context. Below pH 6.0, nitric oxide and nitrate accumulated, and nitrous oxide (N₂O) levels, although negligible compared to the other N-compounds, peaked near pH 4.0. Stable isotope analysis, including the site-specific 15 N-enrichment at the inner (α) and outer (β) nitrogen positions of the N 2 O molecule, indicated nitrifier-denitrification as the source of N₂O, supported by the highest norB expression at this pH. These findings provide new insights into the acid-tolerant physiology of “ Ca . Na. tergens ” and advance its potential application in engineered nitrogen removal systems under acidic conditions. IMPORTANCE The world is facing a climate crisis intensified by human-driven nutrient pollution. Ammonia and the bacteria that oxidize it are central both to the global nitrogen cycle and to wastewater treatment. The acidophilic ammonia oxidizer “ Candidatus Nitrosacidococcus tergens” was previously shown to oxidize ammonia under highly acidic conditions; however, a complete understanding of its metabolism is lacking. Our study now shows that “ Ca . Na. tergens” performs canonical ammonia oxidation across a broad pH range. At pH values below 6, however, a combination of chemical and biological processes leads to the production of nitrate, nitric oxide, and the greenhouse gas nitrous oxide. In addition, we show that these bacteria adapt to proton stress through mechanisms beyond transcriptional mechanisms. Our study highlights the robust metabolism of acidophilic ammonia oxidizers and expands our understanding of nitrification under acidic conditions.

Abstract ‘ Candidatus Liberibacter solanacearum’ (Lso) is a devastating bacterial pathogen of crops transmitted by psyllids. Previously, a link between changes in ubiquitination and the progression of diseases caused by Lso was established. Among the Lso haplotypes, LsoA and LsoB infect solanaceous crops. LsoA induces chlorosis and stunting in tomato, while LsoB infection leads to more severe symptoms, including plant death. To better characterize the molecular basis of the differences in the diseases caused by these haplotypes, we cataloged the ubiquitinome of tomato leaves four weeks after LsoA and LsoB-infection and following infestation with Lso-free psyllids. Our analysis revealed haplotype-specific changes in the plant ubiquitinome. Gene Ontology (GO) terms related to glucose metabolism and proteasome-mediated ubiquitin-dependent protein catabolism were enriched among the differentially ubiquitinated proteins between LsoA-infected and Lso-free infested plants. Additionally, a KEGG pathway analysis determined an overrepresentation of proteins involved in carbon metabolism and fixation via the Calvin cycle. In contrast, the comparison of LsoB-infected and Lso-free infested plants revealed a broader range of enriched GO terms and KEGG pathways, including those associated with carbohydrate and energy metabolism, proteasome activity, and oxidative stress. These results contribute to our understanding of the development of Liberibacter-related diseases and reveal the importance of ubiquitin-related pathways in Lso infection. They also contribute to the expanding inventory of ubiquitinated proteins.