Publications

4366

ABSTRACT Huanglongbing (HLB) is a bacterial disease that affects citrus trees and is considered the most severe citrus disease in the world. The three HLB-associated “ Ca . Liberibacter” species harbor prophage regions which have been described to play critical roles in their evolution and biology. In this study, using infected insect vectors, we assembled and characterized the accessory genome of the first circular de novo “ Candidatus Liberibacter asiaticus” (CLas) assembly (V1R1) from Réunion, one of the sparse areas worldwide hosting CLas and “ Ca . Liberibacter africanus” (CLaf). This 1.272 Mb-long whole-genome harbored 1,129 coding sequences and two complete prophages, including a 37,934 bp-long Type 1 prophage, frequently present in CLas genomes, and a 40,501 bp-long undescribed CLas prophage designated as P-V1R1-5. Comparative genomic approaches suggested that P-V1R1-5 has all the genetic elements to produce new viral particles and revealed that it likely belongs to a new CLas Type 5 prophage. While P-V1R1-5-like prophages had been identified previously in CLaf genomes, our study demonstrates the first lysogenic conversion of these prophages in CLas, suggesting a positive selection of prophage-like sequences among HLB-associated “ Ca . Liberibacter” species. Using PCR amplifications targeting P-V1R1-5, we also showed that 85.7% of the tested CLas strains from Réunion and all tested CLaf strains from Madagascar and Réunion harbored a Type 5-like prophage. The identification of CLas Type 5-like prophages not only expanded our knowledge of CLas genomic diversity in Réunion but also provided new insights into the role of horizontally transferred elements in the evolution of the sympatric HLB-associated bacteria. IMPORTANCE Huanglongbing (HLB) is the most severe citrus disease worldwide. The disease is associated with three “ Candidatus Liberibacter” species harboring prophage regions that have been described to play critical roles in their biology. In this study, using infected insect vectors, we assembled and characterized the accessory genome of the first circular de novo “ Ca . Liberibacter asiaticus” (CLas) assembly from Réunion, one of the very few areas in the world where both of CLas and “ Ca . Liberibacter africanus” (CLaf) coexist. Comparative genomic approaches demonstrated that this genome harbored two complete prophages, including a new CLas Type 5 prophage that was previously identified in CLaf but was reported for the first time in a CLas strain. This first evidence of shared prophage-like sequences among HLB-associated species expands our knowledge of CLas genomic diversity but also provides new insights into the role of the accessory genome in the evolution of these bacteria.

Abstract Background Hemotropic Mycoplasma spp. (hemoplasmas) parasitize erythrocytes and cause hemolytic anemia in several mammalian species, including cats. Mycoplasma haemofelis ( Mhf ), “ Candidatus Mycoplasma haemominutum” ( C Mhm) and “ Candidatus Mycoplasma turicensis” ( C Mt) are the three main feline hemoplasma species. A species closely related to C Mhm was recently proposed as a putative novel species based on the 23S ribosomal RNA (rRNA) gene. Methods In this study, 16S and 23S rRNA genes were used to investigate hemoplasma diversity in cats. Blood samples from 388 cats were obtained and screened for hemoplasma infection based on a PCR assay targeting the 16S rRNA gene. Positive samples were sequenced based on the 16S and 23S rRNA genes. All obtained sequences were analyzed by the nucleotide Basic Local Alignment Search Tool (BLASTn), the DnaSP6 computer program, phylogenetic construction, genetic network and pairwise identity matrix. Results The 388 blood samples collected from the cats were screened for hemoplasma infection. The tests showed that 68 cats (17.5%, 95% confidence interval [CI] 13.9–21.7%) were positive for hemoplasmas. Of these 68 positive samples, 49 were successfully sequenced for both the 16S and 23S rRNA genes and the sequences subsequently assigned to 11 nucleotide sequence types (ntSTs). The 16S rDNA analysis revealed one Mhf group, at least three groups within C Mhm and at least two groups within C Mt. Notably, we identified C Mhm as well as two putative species closely related to C Mhm from 23S rDNA analysis, including one that has been previously identified. In contrast, the identity of the C Mt-derived 23S rDNA sequence ntST#11 remains unclear due to the lack of C Mt reference sequences, highlighting the need for more comprehensive C Mt data in public databases. Conclusions Our data suggested the presence of two putative species related to C Mhm identified in domestic cats in Thailand. Integrating analyses of independent genetic markers, such as 16S and 23S rRNA genes, would enhance hemoplasma species identification and novel species discovery. Graphical abstract

‘ Candidatus Ferrigenium straubiae’ strain KS (KCTC 25982, DSM 118991) is a neutrophilic, Fe(II)-oxidizing bacterium representing up to 98% of the community in culture KS, the most extensively studied mixed culture known for autotrophic nitrate-reducing Fe(II) oxidation. The phylogeny and genome of this bacterium were previously analysed and identified as ‘ Candidatus Ferrigenium straubiae’. In this study, we report the first-time successful isolation of ‘ Candidatus Ferrigenium straubiae’ strain KS and its experimental physiological characterization. The bacterium was identified as a non-stalk-forming, rod-shaped and non-halophilic strain with a Gram-negative classification. We characterized its physiology when grown in agarose-stabilized Fe(II)-O 2 gradient tubes where Fe(II) stemming from FeS functions as the electron donor and O 2 as the electron acceptor. It showed growth at temperatures of 20–30 °C (optimal at 25°C) and at pH levels of 6.0–7.5 (optimal at pH 6.5–7.0). The doubling time at 20 °C and pH 6.5 was 16 h. We further optimized the gradient tubes for sustainable culture maintenance using modified Wolfe’s mineral medium (MWMM; 1 g l −1 NH 4 Cl) supplemented with 7-vitamin solution, SL-10 trace elements, selenite-tungstate solution and selenite-molybdate-nickel-copper-arsenic-vanadium solution (final concentrations of 10 µM Se, 10 µM Mo, 0.1 µM Ni, 0.1 µM Cu, 0.1 µM As and 5 nM V). We also evaluated several Fe(II) sources (with O 2 as electron acceptor), as well as both inorganic and organic substrates for their influence on growth. Although a known member of the denitrifying community in culture KS, the isolated strain ‘ Candidatus Ferrigenium straubiae’ KS exhibited exclusively microaerobic and autotrophic growth in agarose-stabilized Fe(II)-O 2 gradients, utilizing Fe(II) from FeS as the electron donor.

Abstract The candidate phylum Cloacimonadota is frequently detected in anaerobic environments such as anaerobic digestion (AD) reactors, hydrothermal vents, and deep-sea sediments, yet its metabolism remains poorly understood due to the lack of cultured representatives. Metagenomic evidence suggests capacities for amino acid fermentation, cellulose degradation, and production of carbohydrate-active enzymes, with particular interest in their presumed role in syntrophic propionate oxidation (SPO), a key bottleneck in AD. However, a complete methylmalonyl-CoA (mmc) pathway, central to SPO, has not been previously identified in Cloacimonadota genomes. Here, we report results from a lab-scale anaerobic baffled reactor fed with sugar beet pulp, where a sharp increase in an uncultured Cloacimonadota OTU coincided with recovery of methanogenesis and enhanced methane production. Metagenomic and metatranscriptomic analyses enabled metabolic reconstruction of this OTU, complemented by a curated database of 47 genome-resolved Cloacimonadota species. Comparative genomics revealed conserved protein clusters indicative of an alternative mmc pathway, suggesting that this variant of the SPO pathway is a widespread, phylum-specific trait potentially linked to protein degradation and poly-γ-glutamate biosynthesis. Network analysis identified the methanogenic archaeon Methanothrix as a primary syntrophic partner, an interaction further supported by propionate-fed enrichment cultures showing co-occurrence of Cloacimonadota and Methanothrix species. Our study sheds light on the Cloacimonadota metabolism, advancing our understanding of their ecological roles and potential for biotechnological applications.

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.