Publications

4513

Among citrus diseases, Huanglongbing (HLB) is recognized as the most destructive and economically damaging worldwide. It is mainly associated with ‘ Candidatus Liberibacter asiaticus’ ( C Las) being transmitted by Diaphorina citri . There are no curative treatments or commercial citrus varieties resistant to C Las. Wild Aurantioideae species have been widely screened and, recently, Oceanian genotypes graft- and sexually compatible with Citrus were identified as HLB-resistant; however, there is no information regarding their use as interstocks of commercial varieties yet. Under greenhouse conditions, six HLB-resistant genotypes were evaluated as interstocks between ‘Valencia’ sweet orange scion and ‘Rangpur’ lime rootstock, both susceptible to HLB, with ‘Valencia’ interstock as the control. Rootstocks were nucellar seedlings, and the scion was a commercial accession preimmunized with a mild CTV strain. Plants were assessed for C Las infection and titer in leaves, stem bark, and roots up to 12–24 months after graft-inoculation in the scion and compared to non-inoculated controls. Furthermore, molecular, anatomical, and biometric variables were investigated. As expected, the scion variety was colonized by C Las regardless of the genotype evaluated as interstock. Although bacteria were detected in the roots of most C Las-inoculated plants, C Las movement from the scion to the roots was blocked in 42% and 86% of composite plants when using a F1 hybrid of C. australis × C. inodora or an admixture hybrid of C. glauca , C. australis , and C. australasica as interstocks. Overall, C Las titers were similar in infected plant tissues among the evaluated genotypes, but titers were lower in interstock bark tissues compared to scion and rootstock ones. After one-two years of C Las + CTV infection (experiments I and II, respectively), the dry weight of the root system decreased by 50% in infected trees compared to control trees for most genotypes, and C Las + CTV infection was associated with changes in the sieve phloem and gene expression. These findings suggest that, despite CTV infection, interstocks derived from some hybrids of Australian citrus types have the potential to restrict the movement of C Las from the scion into the roots of infected citrus trees. Long-term evaluation of composite plants in field conditions is necessary to assess tree performance and, ultimately, the impact of C Las blockage by interstocking on HLB disease damage.

Abstract Ferritins are ubiquitous among life forms, as they are essential for iron homeostasis. Here, we unveiled a novel member of the ferritin family, baptised mini-bacterioferritin. The characterised mini-bacterioferritin was isolated from a microbial enrichment dominated by the methanotrophic archaeon ‘ Candidatus Methanoperedens carboxydivorans’. Its atomic resolution crystal structure reveals a 12-mer assembly with a diiron ferroxidase centre located within a four-helix bundle. Redox-cycling experiments on protein crystals reveal a shift in iron position at the active site, which follows the established ferritin catalytic cycle. The 12-mer sphere-like structure harboured six Fe-coproporphyrin III ligands, positioned at the interdimeric interface, a characteristic previously only found in 24-mer bacterioferritins. Phylogenetics, together with structure predictions of closely related proteins, revealed that mini-bacterioferritins form a distinct clade within the ferritin family that might conserve ancestral traits. Future research will need to investigate the physiological roles of these enzymes, which were unsuspectingly widely distributed among prokaryotes.

Abstract Anaerobic methanotrophic (ANME) archaea are important players in the microbial methane cycle, mitigating methane emissions from anoxic environments. ANME are found ubiquitously in methane-rich sediments, where they can couple anaerobic methane oxidation (AOM) to different electron acceptors such as sulfate, metal oxides, and natural organic matter (NOM). However, we still lack understanding of the geochemical niches and preferred metabolic pathways of most ANME subclades. Here, we investigated the genomic potential and ecophysiology of ANME-2a with respect to metal-dependent AOM in brackish metal-rich coastal sediments. We assembled several high-quality ANME MAGs from subclades with high strain heterogeneity and analyzed the genomic potential for metal-AOM. Additionally, we monitored long-term enrichments with various electron acceptors from the same sediments. Ultimately, we recovered 8 novel genomes of ANME-2a that clustered with an uncharacterized genus with only 2 representatives in public databases for which we propose the name ‘ Candidatus Methanoborealis’. The analysis of the MAGs showed two different clusters within this genus; one comprising of MAGs from the Baltic Sea that showed high potential for extracellular electron transfer (EET) required for metal-AOM, and another cluster form more diverse environments with less EET potential. The Baltic Sea Ca. Methanoborealis were the only canonical methanotrophs in the incubations during active methane oxidation and metal reduction. Our results contribute to the understanding of the phylogenomic and metabolic diversity in ANME subclades, which will help to further characterize novel ANME lineages from complex sediment samples.

Two species have been named in the provisional genus ‘ Candidatus Nostocoides’: ‘ Ca . Nostocoides limicola’ corrig. Blackall et al . 2000 and ‘ Ca . Nostocoides acidiphilum’ corrig. Kulichevskaya et al . 2012. The type species of the genus, ‘ Ca . Nostocoides limicola’ has since been transferred to Tetrasphaera Maszenan et al . 2000, which was later renamed Nostocoides Deshmukh and Oren 2024. This leaves ‘ Ca . Nostocoides acidiphilum’ as an orphan species. Furthermore, Kulichevskaya et al . did not assign ‘ Ca . Nostocoides acidiphilum’ to ‘ Ca . Nostocoides’ corrig. Blackall et al . 2000. Instead, they proposed a new genus, ‘ Ca . Nostocoides’ corrig. Kulichevskaya et al . 2012, in the phylum Planctomycetota . This is in violation of Rule 51b (4) of the International Code of Nomenclature of Prokaryotes, making ‘ Ca . Nostocoides’ corrig. Kulichevskaya et al . 2012 and ‘ Ca . Nostocoides acidiphilum’ corrig. Kulichevskaya et al . 2012 pro-illegitimate names. To resolve this situation, I propose the replacement names ‘ Ca . Nostocimimus’ gen. nov. and ‘ Ca . Nostocimimus acidiphilus’ comb. nov., respectively.

Abstract Grapevine plants showing symptoms typically induced by phytoplasmas infection, characterized by leaf reddening or yellowing, reduce vigour, delayed growth and shoot proliferation with reduced leaf size, were observed in the state of São Paulo, Brazil. This disorder causes significant damage to grape production by delaying the onset of fruit bearing and reducing yield. Similar symptoms have been reported in grapevines grown in other countries revealing the presence of phytoplasmas in diseased plants. Based on the described symptoms, this study was conducted in order to demonstrate the association between phytoplasma and symptomatic grapevine plants, as well as characterize molecularly the detected pathogen. Molecular analysis was performed using PCR and RFLP assays, followed by phylogenetic analysis. Amplifications of genomic fragments of 1.2 evidenced the presence of phytoplasma in tissue of symptomatic samples confirming the preliminary diagnosis based on the symptoms. Molecular characterisation identified the phytoplasma as a ‘ Ca Phytoplasma prunorum’ strain, belonging to the 16SrX-B subgroup. This is the first report of a phytoplasma belonging to the 16SrX-B group infecting grapevine plants in Brazil.

Abstract The candidate phylum Cloacimonadota is frequently detected in anoxic environments such as anaerobic digestion (AD) reactors, hydrothermal vents, and deep-sea sediments, yet its metabolism remains poorly understood. Metagenomic evidence suggests capacities for amino acid fermentation, carbohydrate degradation, as well as a potential 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 an acidified lab-scale anaerobic baffled reactor fed with sugar beet pulp, where an increase in the relative abundance of Cloacimonadota correlated with recovery of methanogenesis, resulting in increased methane content in the produced biogas. Metagenomic and metatranscriptomic analyses enabled metabolic reconstruction of the dominant Cloacimonadota OTU. Furthermore, using a curated database of 204 genome-resolved Cloacimonadota species, we characterised the phylum-level metabolic potential. Comparative genomics revealed alternative proteins, including 2-oxoglutarate:ferredoxin oxidoreductase and aspartate aminotransferase, likely to substitute for missing enzymes in the classical mmc pathway. These proteins were widely distributed and highly conserved across the analysed Cloacimonadota genomes, suggesting that this variant of the SPO pathway could represent a phylum-specific trait. Moreover, we hypothesise that these alternative pathway steps may link propionate metabolism to protein degradation and poly-γ-glutamate biosynthesis. Network analysis identified the methanogenic archaeon Methanothrix as a potential 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.

Abstract Hemotropic mycoplasmas (hemoplasmas) infecting cattle were first detected in France in 2019 during a clinical outbreak, but their prevalence both at the species and strain levels remains unknown. This study therefore aimed to determine the prevalence of hemoplasmas and their coinfections, and to identify associated risk factors using a fixed-effect logistic regression model. Detection was performed using 16S rRNA qPCR, as well as three strain-specific rnpB qPCR assays targeting Candidatus Mycoplasma haematobovis (CMh, formerly Ca . M. haemobos), Mycoplasma wenyonii strain Mexico (Mex), and Mycoplasma wenyonii strain Massachusetts (Mass). A total of 1011 cattle were sampled, of which 88.7% (897/1011) tested positive by 16S rRNA qPCR. Prevalence rates for CMh, Mex, and Mass were 67.7% (684/1011), 65.9% (666/1011), and 19.8% (200/1011), respectively. Concordance between 16S rRNA qPCR and strain-specific rnpB qPCR was 92% (931/1011). Coinfections were detected in 67.8% (565/833) of infected animals, with the CMh + Mex combination being the most frequent. The Mass strain was predominantly found in triple infections. Risk factors for infection included coinfections, age (except for Mass), and climatic conditions for CMh and Mass. Further studies are needed to elucidate transmission routes and pathogenicity mechanisms of hemoplasmas.

Abstract Cassava is a key food and energy crop in the tropical and subtropical regions. In South America, oversprouting disease (OVSPD) and cassava frogskin disease (CFSD) occur frequently, however, their etiologies remain unknown. OVSPD is characterized by excessive shoot proliferation, chlorosis, and dwarfism, whereas CFSD largely affects roots, causing cork-like tissues with honeycomb patterns and reduced starch content. In Brazil, phytoplasma organisms were found to be associated with both diseases, with sequences of the 16SrIII-B subgroup phytoplasmas linked to OVSPD and the 16SrIII-L and 16SrIII-A subgroups linked to CFSD. To clarify the identity and taxonomic position of the putative phytoplasma in the diseases, high-throughput sequencing (HTS) of total plant DNA from cassava plants with OVSPD and others with CFSD symptoms was performed, and the complete genome of a novel phytoplasma was assembled from OVSPD plants. In contrast, HTS from CFSD affected plants revealed short sequence fragments of diverse phytoplasmas, however, the assembly of larger scaffolds failed. Genome analyses of two OVSPD phytoplasma assemblies confirmed a novel phytoplasma species within the 16SrIII group. It has an average nucleotide identity (ANI), below the 95% species demarcation threshold relative to the genomes of other phytoplasma species and we propose the name ' Candidatus Phytoplasma manihotii' sp. nov. A broader screening showed that this phytoplasma occurs only in OVSPD plants, suggesting that the genome fragments detected in CFSD plants may have resulted from other sources, such as genomic integration or horizontal gene transfer.

Desmodium styracifolium (Osb.) Merr., a member of the Leguminosae family, is an important medicinal plant widely used in traditional Chinese medicine. In September 2024, D. styracifolium plants exhibiting symptoms of little leaf and stunted growth were observed in a field of Zhanjiang, Guangdong province, China. Since the symptoms resembled those associated with phytoplasma infections, total DNA was extracted from the leaves of four symptomatic plants and one healthy plant for molecular identification. Universal primer pairs (P1/P7, R16mF2/mR1) for phytoplasma detection were used to amplify the 16S rDNA fragments (~1.8 kb and ~1.4 kb), while a specific primer pair secY-F/secY-R was employed to amplify a ~1.4 kb segment of the secY gene. Target fragments were successfully amplified from all symptomatic samples but not from the healthy control. These amplicons were cloned and sequenced. The obtained 16S rDNA sequence of D. styracifolium little leaf phytoplasma (DsLFP-GDZJ) showed the highest identity (99.67–100%) with strains of ‘Ca. Phytoplasma australasiaticum’ (subgroup 16SrII-A and 16SrII-D). Phylogenetic analysis also indicated that DsLFP-GDZJ formed a small evolutionary branch with strains of ‘Ca. Phytoplasma australasiaticum’ (subgroup 16SrII-A and 16SrII-D). Virtual RFLP (restriction fragment length polymorphism) analysis of the 16S rDNA sequence demonstrated DsLFP-GDZJ belongs to the 16SrII-A subgroup (GenBank accession number L33765). The secY gene sequence of DsLFP-GDZJ also showed the highest similarity and the closest relationship with those of the 16SrII-A subgroup phytoplasma strains. These results showed that DsLFP-GDZJ is a strain of ‘Ca. Phytoplasma australasiaticum’ (16SrII-A subgroup). To our knowledge, this is the first report of ‘Ca. Phytoplasma australasiaticum’—related phytoplasma associated with D. styracifolium little leaf disease in China, thereby establishing D. styracifolium (Osb.) Merr. as a new host plant of phytoplasma.

The Asian citrus psyllid, Diaphorina citri, transmits the putative causal agent of citrus greening disease, ‘Candidatus Liberibacter asiaticus’. The transmission occurs in a propagative circulative manner. Specific protein-protein interactions are required for the accomplishment of the transmission process. We employed the far-Western technique, also known as protein overlay assay, and mass spectrometry, to identify D. citri proteins that specifically recognize membrane proteins of ‘Ca. L. asiaticus’. Whole-body protein extracts of D. citri were separated by electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were overlayed with sonicated leaf midrib extracts from ‘Ca. L. asiaticus’-infected citrus plants. The protein complexes were detected using different antibodies against ‘Ca. L. asiaticus’ membrane proteins and peptidoglycans associated with lipoproteins. By comparison, spots were extracted from a polyacrylamide gel for LC-MS-MS analysis. Identified proteins included ATP synthase alpha and beta subunits, actin, alpha- and beta-tubulins, transitional endoplasmic reticulum ATPase TER94, 78 kDa glucose-regulated protein, and arginine kinase. These findings emphasized the involvement of energy machinery and muscle proteins in the ‘Ca. L. asiaticus’-D. citri interactions. Understanding the pathogen-vector interactions will lead to better-designed control strategies based on the interference with specific interactions and/or blocking transmission.