Publications

4511

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, and 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 2 potential mobile units. Analyses of published peanut and sweet potato phytoplasma genomes revealed 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 pangenome analysis 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 of the genome. This study provides valuable genomic insights into this phytoplasma, characterizing 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.

Shorea assamica is an evergreen to semi-evergreen tree in the family Dipterocarpaceae. It is widely distributed in China, India, Myanmar, Malaysia, Indonesia, the Philippines, and other regions. In China, it is found in western Yunnan and southeastern Tibet. The species is valued for its aromatic white resin, which serves as an important chemical raw material (Ang and Maruyama 1997). In May 2025, S. assamica trees exhibiting symptoms of witches'-broom disease and plexus bud disease were found at Mengmao, WanDing, Nongdao, Jiegao, and Jiexiang in RuiLi County (97°31′-98°02′ E, 23°38′-24°14′ N), Dehong Prefecture, Yunnan Province, China. Following disease onset, the overall growth of infected plants was significantly slower compared to healthy individuals. Infected plants exhibited excessive branching, shortened internodes, and a clustered growth symptom. Lateral buds on diseased tree frequently developed into abnormal twigs, with many buds forming dense clusters and only a few producing leaves. The disease was designated as S. assamica witches'-broom (SAWB). Across the seven surveyed areas, over 74% of trees displayed symptoms, while the disease incidence in nursery-cultivated seedlings was 66%. The disease was found to be spreading actively. A total of 56 samples were collected from seven areas, including 42 symptomatic plants and 14 asymptomatic plants. The lateral stem tissues of 30 samples were observed under a scanning electron microscope (Hitachi S-3000N). Spherical bodies were found in the phloem sieve cells of symptomatic plants. Total DNA was extracted from 56 plant samples using the CTAB method (Porebski et al. 1997) and subjected to nested PCR testing. Double-distilled water was used as negative control, and the DNA extracted from Dodonaea viscosa affected by D. viscosa witches’-broom disease was used as a positive control. Nested PCR was employed to amplify the pathogen’s 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993), and 1.2 kb segment was produced (GenBank accessions: PZ025091, PZ025092, and PZ025093). PCR specific to the ribosomal protein (rp) gene yielded a segment of approximately 1.2 kb (Lee et al. 2003) (GenBank accessions: PZ028767, PZ028768, and PZ028769). The fragment size from 31 samples was consistent with the positive control, confirming the association of phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of S. assamica witches’-broom phytoplasma showed that it shared 99.68% identity with that of Rice orange leaf phytoplasma (GenBank accession: JQ965690). The rp sequence shared 99.83% identity with that of Salix tetradenia witches'-broom phytoplasma (GenBank accession: KC117314). An analysis with iPhyClassifier (Zhao et al. 2013) showed that the virtual RFLP pattern derived from the 16S rDNA fragment of SAWB phytoplasma was identical (similarity coefficient 0.98) to the reference pattern of 16Sr group I, subgroup B (OY-M, GenBank accession AP006628). The SAWB phytoplasma is a variant of 16SrI-B. The phylogenetic tree was reconstructed based on 16S rRNA and rp gene sequences using MEGA (Tamura et al. 2013). The analysis was conducted using the neighbor-joining method with 1,000 replicates of bootstrap analysis. The results indicated that the SAWB phytoplasmas grouped into clades including phytoplasmas belonging to 16SrI-B and rpI-B, respectively. In addition, 1-year-old S. assamica were used for grafting assays in nursery, the twigs from infected S. assamica under natural conditions were used as a scion, and the phytoplasma was detected using nested PCR after grafting for 40 days. To the best of our knowledge, S. assamica is a new host of ‘Ca. P. asteris’-related strain (16SrI-B) in China. The newly emerged disease is a threat to S. assamica.

Abstract Sulfoquinovose, a sulfonated sugar derived from the thylakoid membrane lipid sulfoquinovosyl diacylglycerol, is abundant in photosynthetic organisms and plays a key role in global sulfur cycling. Its degradation in nature is mediated by specialized bacteria, many of which rely on the enzyme sulfoquinovosidase (YihQ) to release sulfoquinovose from sulfoquinovosyl (diacyl)glycerol. Despite its ecological importance, the diversity and functional roles of sulfoquinovose-degrading microorganisms remain poorly characterized in natural environments. Here, we developed a yihQ-targeted amplicon sequencing approach to investigate the richness and distribution of SQ-degrading bacteria across selected environments. We revealed high richness of yihQ-containing microorganisms in the analyzed cow rumen samples, far exceeding that observed in human and mouse gut microbiomes, suggesting an important role of sulfoquinovose metabolism in ruminant digestion. Anoxic microcosm experiments with sulfoquinovose-amended rumen fluid revealed cooperative microbial degradation of sulfoquinovose to sulfide via isethionate cross-feeding. Amplicon sequencing and genome-resolved metagenomics and metatranscriptomics identified yet undescribed and uncultured sulfoquinovose-degrading taxa. Members of Caproiciproducens (Acutalibacteraceae), Candidatus Limivicinus (Oscillospiraceae), and Sphaerochaetaceae transcribed the isethionate-producing sulfo-transketolase pathway, whereas isethionate was likely respired by a Candidatus Mailhella bacterium (Desulfovibrionaceae). This study presents a functional gene-based assay for tracking environmental yihQ richness, highlights sulfoquinovose degradation as a central metabolic process in the cow rumen, describes previously unknown sulfoquinovose-metabolizing bacteria, and advances understanding of sulfur physiology in complex microbial communities.

‘Candidatus Liberibacter solanacearum’ (Lso) haplotype D, transmitted by the carrot psyllid Bactericera trigonica, is a major threat to carrot production, particularly in the Mediterranean Basin and the Middle East. We investigated the impact of plant age at the time of inoculation on symptom development and yield under greenhouse and outdoor conditions. In greenhouse trials, 1- and 2-month-old plants were inoculated with psyllids harboring Lso. Both age groups showed reduced taproot weight compared to controls, but younger plants were more severely affected, exhibiting 90% yield reduction, while 2-month-old plants showed a 30% reduction. Witches’ broom symptoms appeared earlier in plants inoculated at 1-month-old (5 weeks post-inoculation); however, within two weeks, plants inoculated at two months reached comparable levels of symptom incidence and severity. Additional greenhouse experiments using Lso-free psyllids confirmed that yield losses and disease symptoms were specifically due to Lso infection, and not psyllid feeding. In outdoor trials, we tested the effects of inoculation at 7, 11, 15, and 19 weeks post-sowing (wps) on disease incidence and yield. Significant yield losses occurred only in plants inoculated at 7 and 11 wps, with up to 45% reduction by harvest time. Symptoms began to appear at 20 wps in plants inoculated at 7 and 11 wps. At 23 wps, symptoms also appeared in plants inoculated at 15 wps, with a similar incidence to that observed in the earlier inoculation groups. Molecular detection of Lso at harvest (23 wps) confirmed that all plants inoculated at 19 wps or earlier were infected. These results demonstrate that carrots are highly susceptible to Lso-induced yield losses at the earlier stages of growth and highlight the importance of managing early-season vector pressure to minimize economic damage.

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 We report the draft metagenome-assembled genome (MAG) of “ Candidatus Phytoplasma australasiaticum” strain TBB-AP, obtained from a symptomatic tomato plant collected in Andhra Pradesh, India. This assembly provides a genomic resource for functional and evolutionary studies of phytoplasmas associated with tomato big bud disease.

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.