Publications

4391

Abstract Background Globally, the disease ecology of reptiles remains understudied, even for threatened and iconic species such as the Galápagos marine iguana ( Amblyrhynchus cristatus ). Although marine iguanas are parasitized by distinct species of ticks and mites, research on vector-borne diseases for this species is limited. Methods In this study, we detected 16S ribosomal RNA (rRNA) sequences of Candidatus Allocryptoplasma in transcriptomic data from marine iguana blood samples. These 16S rRNA sequences were further characterized through phylogenetic analysis and a haplotype network. Results Our analysis revealed the first molecular evidence for the infection of marine iguanas with Candidatus Allocryptoplasma, a candidate genus in the family Anaplasmataceae with unknown pathogenic potential, likely transmitted by ticks. Phylogenetic analysis of the novel 16S rRNA sequences together with available Anaplasmataceae sequences confirmed their assignment to this candidate genus. A haplotype network analysis indicated that the agent infecting the marine iguana represents a distinct lineage within the known Ca . Allocryptoplasma diversity. Conclusions Candidatus Allocryptoplasma had a high prevalence within marine iguanas, infecting individuals across most of the geographical range of this species. To elucidate the transmission dynamics of this bacterium in the Galápagos ecosystem, ectoparasites of the marine iguana and shared vertebrate hosts should be screened for infection with Ca . Allocryptoplasma. Graphical Abstract

Abstract The DPANN superphylum is a deep-branching radiation of Archaea with small cell and genome sizes. Most DPANN lineages are predicted or validated to be host-dependent. However, certain lineages have substantial biosynthetic capacities and are potentially less dependent on hosts or even free-living. Here, we reconstructed 163 Micrarchaeota genomes, comprising 48 assigned to previously undescribed orders and 115 affiliated with known orders. Investigation of their genetic repertoire revealed substantial metabolic capacity in Norongarragalinales, Anstonellales, and the newly proposed Wunengiarchaeales-associated lineages, including complete or near-complete glycolysis and de novo biosynthetic pathways for nucleotides, amino acids, co-factors, and cell envelopes. We classified these genes related to central metabolism, but which are uncommon in DPANN archaea as putative free-living associated genes (pFLAGs). The extensive presence of pFLAGs in Norongarragalinales suggests a potential host-independent lifestyle. Reconstruction of evolutionary history revealed that these pFLAGs were not ancestral within the DPANN superphylum. Instead, we suggest that less host-dependent organisms evolved from symbionts through the gradual acquisition of pFLAGs through horizontal gene transfer, whereas other Micrarchaeota lineages with streamlined genomes experienced reductive evolution due to thermal adaptation. Our analyses demonstrate that host dependency is not always an evolutionary dead end, but can be reversed through acquisition of new metabolic capabilities by horizontal transfer.

Phloem feeders, such as the psyllid Diaphorina citri ( D. citri ), feed on plants by inserting their stylet bundle followed by probing the apoplast before reaching the phloem. The psyllids secrete watery saliva containing various proteins into the phloem, which can act as effectors to facilitate their feeding or modulate host defense responses. Concomitantly, feeding is the main mode of transmitting the Candidatus Liberibacter asiaticus ( C Las) bacteria to the phloem. C Las produces several effectors that have been hypothesized to contribute to Huanglongbing (HLB) virulence. Here, we aimed to identify putative effector proteins from both D. citri and C Las. To achieve this, we used different omics techniques on different tissues and organs from both plants and insects. More specifically, we performed transcriptomics on the heads of healthy and C Las-infected D. citri and proteomics of artificial diet and of phloem of four different plant species fed on by healthy and C Las-infected D. citri. Subsequently, we used various criteria and bioinformatics tools to predict putative effectors. This resulted in the identification of four proteins from D. citri [ferritin, prisilkin, CG31997-PA, and pterin-4-alpha-carbinolamine dehydratase-like protein (PCBD)] and two Sec-dependent effectors from C Las, CLIBASIA_04560 and CLIBASIA_05320, that were used for further functional studies. The expression of these six proteins in Nicotiana benthamiana modified the ROS burst triggered by flagellin, indicating that they can indeed function as effector proteins in planta . In addition, expression of the psyllid effectors in planta significantly reduced the growth of Pseudomonas syringae pv. tabaci ( Pta ).

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 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

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.