Publications

4368

Symptoms of growth abnormalities including shoot proliferation, leaf rosetting and witches’ broom symptoms, reduction in leaf size, downward rolling of leaf margins, distortion and chlorotic foliage were observed in several tree species in a Hungarian park forest in Martonvásár in the summer of 2022. In subsequent years, we observed the progression of the disease. This was manifested by the death of shoots and twigs, as well as an overall reduction in growth. The affected woody species were: Fraxinus excelsior, Acer campestre, Acer platanoides, Ulmus minor and Sambucus nigra. The observed symptoms were reminiscent of those observed in phytoplasma infections of other woody plants. The present study aimed to investigate whether the disease in these species was also caused by a phytoplasma. Shoot samples were taken from ten symptomatic, and three asymptomatic plants of each species. Total nucleic acids were extracted from the phloem tissue using the CTAB method (Ahrens and Seemüller 1992). To specifically amplify phytoplasma DNA, the universal primer pairs fP1/rP7 (Smart et al., 1996), followed by R16F2n/R16R2 (Gundersen and Lee, 1996)or fU5/rU3 (Lorenz et al., 1995) were used in a nested PCR. Amplicons of the expected sizes were obtained from 90% of the symptomatic samples (fP1/rP7 1783 bp, R16F2n/R16R2: 1.2 kb, fU5/rU3: 876 bp), but none from the asymptomatic ones. The R16F2n/R16R2-amplified products obtained from the five species were cloned (at least three from each PCR reaction), sequenced and deposited in GenBank under the following accession numbers PX491300 (F. exelsior), PX491301 (S. nigra), PX491302 (A. campestre), PX491303 (U. minor) and PX491304 (A. platanoides). The sequences of the five isolates were almost identical, showing a 99.92%-100% identity with each other. A BLAST search of GenBank yielded 99.92% sequence similarity in the case of the S. nigra isolate and 100% in the case of the other 4 isolates with the onion yellows phytoplasma strain OY-M (GenBank AP006628) from the ‘Candidatus Phytoplasma asteris’ 16SrI-B subgroup. To confirm the pathogen’s identity in subsequent experiments, the elongation factor Tu (tuf) and the translocase protein (secY) genes were amplified using the universal primer pairs fTuf1/rTuf1 (Schneider and Gibb, 1997), and the aster yellows-specific AYsecY_F-46/AYsecY_R1450 primer pairs (Viczián et al. 2023), respectively. Amplicons of the expected sizes (1085 bp for fTuf1/rTuf1, and 1520 bp for AYsecY_F-46/AYsecY_R1450) were produced from 84% of the symptomatic plants across all five species, but not from the asymptomatic ones. The PCR fragments were cloned and sequenced, and sequences obtained for the tuf and secY genes were deposited in GenBank under the accession numbers PV648950-PV648954, and PV854856-PV854860, respectively. The sequences obtained from the five plant species were almost identical, sharing 99.81% identity with the Aster yellows phytoplasma strains IRap (AJ271316.1) and ORN (MN526022.1) in the case of the elongation factor TU, and 100% identity with the strains De Villa (CP035949.1) and M8 (CP128414.1) in the case of the SecY translocase protein. In iPhyClassifier analysis, the virtual RFLP patterns derived from the query 16S rDNA F2nR2 fragments were identical (similarity coeffcient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). These results clearly indicate that the five tree species under study were affected by the aster yellows phytoplasma, which is assigned to the 16SrI-B subgroup of the 16SrI group and is caused by the bacterial agent 'Candidatus Phytoplasma asteris'. Although this phytoplasma is known to infect a wide range of hosts (Lee et al., 2004), aster yellows has only been reported on F. exelsior in Poland (Kamińska and Berniak, 2009) of the five plant species studied here. To our knowledge, this is the first report of aster yellows on A. campestre, A. platanoides, U. minor and S. nigra worldwide, and on F. exelsior in Hungary.

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.