Publications

4954

ABSTRACT Plant microbiomes are a rich source of secondary metabolites, often with pathogen- or disease-suppressive traits. Huanglongbing (HLB) is a serious disease of citrus for which there is currently no cure. Here, we tested the hypothesis that bacteria native to the citrus microbiome may possess inhibitory activity against the associated causal agent of HLB, Candidatus Liberibacter asiaticus ( C Las). We coupled a robotic microfluidic isolation platform with a high-throughput antimicrobial screening assay using Liberibacter crescens , a culturable surrogate for the unculturable C Las, to identify isolates with anti- Liberibacter activity. We identified 35 bacterial isolates with activity using this pipeline. One strongly inhibitory citrus root isolate, Pseudomonas chlororaphis (SRAM11), was selected for bioassay-guided fractionation. We determined a significant driver of in vitro Liberibacter inhibition was attributed to the production of phenazine-1-carboxamide (PCN) by P. chlororaphis . We also demonstrated significant anti- C Las activity of PCN, comparable to that of oxytetracycline, using an ex vivo citrus hairy root assay. P. chlororaphis and phenazines have been implicated in successful biocontrol of other diseases and represent a new tool with possible application to HLB mitigation. IMPORTANCE Huanglongbing (HLB) is a devastating citrus disease that causes millions of dollars in crop loss each year in the United States alone. In this study, we mined the citrus microbiome for bacteria that produce antimicrobials with suppressive activity against Candidatus Liberibacter asiaticus, the causal agent of HLB. We demonstrated that the citrus microbiome is a rich source of microorganisms that inhibit Liberibacter spp. We selected a Pseudomonas chlororaphis strain for further characterization and assigned anti- Liberibacter activity to the production of phenazine-1-carboxamide. Phenazine-producing P. chlororaphis strains are known biocontrols in other pathosystems, and this study highlights their potential for mitigation of HLB.

A novel phytoplasma species, ‘ Candidatus Phytoplasma zeae’, is proposed based on ecological distinctiveness, vector specificity, whole-genome comparisons and community consensus. This phytoplasma is associated with maize bushy stunt (MBS) disease in corn ( Zea mays ) and is transmitted exclusively by Dalbulus maidis and Dalbulus elimatus , two leafhopper species endemic to the Americas that has been reported in Brazil, Colombia, Mexico, Peru and several U.S. states. Here, we sequenced and assembled the genome of MBS phytoplasma (MBSP) strains from Brazil and the USA to describe and propose this new species. Although the 16S rRNA gene sequence of the proposed reference strain, MBSP-BR RS , shares >99% identity with that of ‘ Candidatus Phytoplasma asteris’, key nucleotide polymorphisms distinguish ‘ Ca . P. zeae’ from other 16SrI-related phytoplasma species. Genome-wide average nucleotide identity between ‘ Ca . P. zeae’ and all available ‘ Ca . P. asteris’-related strains genomes was between 92.6 and 98.4%. Comparative genomic analyses revealed unique gene clusters in ‘ Ca . P. zeae’ associated with amino acid transport, defence mechanisms and protein turnover, which may contribute to its specialization in corn. The ecological profile of ‘ Ca . P. zeae’, including its narrow host and vector range and restricted geographic distribution, supports its recognition as a novel species under Rule c of the IRPCM guidelines. The designation ‘ Ca . P. zeae’ is, therefore, proposed by members of the research community who have studied this pathogen for over a decade, with the MBSP-Brazil RS strain serving as the reference.

Abstract Enhanced biological phosphorus removal (EBPR) relies on polyphosphate-accumulating organisms that experience dynamic fluctuations in redox conditions and substrate availability. How these organisms regulate metabolism across molecular layers under cyclic operation remains incompletely understood. Here, we investigated molecular regulation in a long-term, stable aerobic granular sludge system dominated by Candidatus Accumulibacter . We combined metagenomics, time-resolved metatranscriptomics, and matched metaproteomics across three consecutive operational cycles. Genome-resolved metagenomics revealed an Accumulibacter -dominated community encoding canonical EBPR functions. Time-resolved metatranscriptomics revealed extensive and reproducible transcriptional dynamics affecting hundreds of genes. These dynamics did not align with discrete EBPR phases or instantaneous environmental transitions, but instead unfolded gradually over the cycle. Despite pronounced transcript-level heterogeneity, aggregation at the functional level revealed strong consolidation, with only a limited number of KEGG orthologs exhibiting coherent temporal regulation. Quantitative metaproteomics revealed a stable proteome across EBPR cycles, with no individual proteins or functional categories showing significant differential abundance despite direct transcript-protein mapping and time-matched sampling. This indicated that transcriptional dynamics did not translate into measurable changes in protein pool size on the time scale of individual cycles, consistent with slow, growth-coupled proteome turnover relative to cycle duration. Together, these results support a hierarchical model of regulation in Candidatus Accumulibacter , in which dynamic transcriptional responses are buffered at both the functional and protein levels. This organization provides a plausible basis for stable EBPR performance under predictable cyclic operation and demonstrates how time-resolved multi-omics can be used to resolve regulatory dynamics across molecular layers in engineered microbial ecosystems.

Abstract In northeastern Argentina, wild canids such as the pampas fox ( Lycalopex gymnocercus ) and the crab‐eating fox ( Cerdocyon thous ) coexist with domestic dogs ( Canis lupus familiaris ) across rural and peri‐urban landscapes, creating opportunities for tick and pathogen exchange. Characterizing tick assemblages and associated pathogens in these hosts is essential to assess potential public health risks at the wildlife–domestic interface. Ticks were collected opportunistically from domestic dogs and road‐killed foxes near two protected areas in Corrientes Province. Specimens were identified using morphological and molecular approaches and screened for Rickettsia spp. by PCR. A total of 30 adult ticks were obtained from 12 dogs and 8 foxes, representing four species: Amblyomma tigrinum , A. ovale , A. dubitatum and Rhipicephalus sanguineus . Amblyomma tigrinum was the most frequent species (50%). Seven individual ticks were confirmed as Candidatus Rickettsia andeanae in A. tigrinum , while one pooled sample was positive by screening only and was not confirmed. These findings expand the known geographic distribution of Ca. R. andeanae and provide molecular evidence of its circulation in ticks collected from both wild and domestic canids in northeastern Argentina. Our results highlight the importance of these hosts as sentinels for tick‐borne pathogens and underscore the need for integrated One Health surveillance to better anticipate emerging potential risks in interface areas.

Huanglongbing (HLB) is one of the most devastating diseases of citrus nowadays. The etiological agent is a fastidious and non-culturable bacterium known as Candidatus Liberibacter asiaticus ( C Las). C Las is a phloem-restricted pathogen, which makes the efficient delivery of antimicrobial compounds to control it a major challenge for citriculture. Therefore, the development of formulations capable of penetrating plant barriers and reducing the bacterial population within the phloem is critical for the control of HLB. Here we report on the development of a novel formulation loaded with oregano essential oil, hexyl gallate and eugenol, described in the literature as a transdermal permeation enhancer. The most effective formula was selected in vitro against bacterial surrogates ( Rhizobium tropici and Rhizobium phaseoli ), displaying bactericidal concentrations of 0.25 and 0.5% (v/v), respectively, of bactericidal concentration. The selected formulation was characterized as a near-Newtonian fluid, with 98.5% of particles measuring around 50 nm, a Polydispersity Index of 0.31, and zeta potential of -17.5 mV. Furthermore, Scanning electron microscopy revealed the presence of nanoparticles, alongside microscopic aggregates. The formulation maintained its bactericidal activity over 120-day storage period. During the root-drenching assays, the formulation significantly reduced overall C Las titres by 1.29 and 2.46 log 10 over a period of 300 days, in two independent experiments. Furthermore, ecotoxicological assessments revealed no acute toxicity in Tenebrio molitor or significant effects in the survival of Melipona scutellaris under the tested conditions. Our findings demonstrate the potential of this non-synthetic formulation as an effective and safe alternative for HLB management, without disrupting non-target organisms.

‘ Candidatus Liberibacter asiaticus’ ( C Las) is a phloem-limited bacterium that causes Huanglongbing (HLB), the devastating citrus greening disease that has led to billions of dollars in losses throughout global citrus production. There is very little epidemiological data available regarding fine-scale molecular variability of C Las populations in different regions of the world or their distribution across various geographical landscapes. Here, we present an assessment of C Las genetic diversity using a hypervariable variable number tandem repeat (VNTR) marker (AGACACA) at the CLIBASIA_01645 locus of 344 genomic sequence data obtained from GenBank from five regions of the world (India-177, China-113, Saudi Arabia-37, Bhutan-10, and Nepal-7). The findings revealed high evolutionary plasticity and non-random distribution of TRN within four distinct classes, significantly deviating from a uniform distribution ( p  < 0.05). The analyzed populations were stratified into four distinct classes: Class I (TRN ≤ 5), Class II (TRN > 5 ≤ 10), Class III (TRN > 10 ≤ 15), and Class IV (TRN > 15). The predominant type of subpopulation for all field samples was intermediate variants, with the highest frequencies observed for TRN6 (25%) and TRN7 (17.15%), possibly indicating genomic stability and/or adaptive advantages of specific tandem repeats. On the other hand, lower frequency rare variants (e.g., TRN17, TRN18, and TRN20 at 0.29%) represent some form of continued low frequency micro evolutional replication slippage and corresponding absence of some individual TRs indicates evidence of local purging forces. Crucially, the Indian C Las populations exhibited significantly greater densities of molecular diversity than populations from China, Saudi, Bhutan, and Nepal examined in this study. The distinct TRN signature profiles of these populations’ subpopulations exhibit strong geographic correlation with ecological regions, reinforcing the CLIBASIA_01645 locus as a strong marker for epidemiological surveillance for tracking global strain movement to develop target-specific HLB management programs.

Abstract The evolution of phototrophy and carbon fixation in the phylum Chloroflexota remains incompletely understood, largely due to the scarcity of cultured mesophilic representatives. Here, we report the isolation and genomic characterization of a novel filamentous anoxygenic phototrophic bacterium from a microbial mat in the mesothermal Lake Umkhey (Southern Siberia). Although the original culture was lost, its genome was recovered via metagenome-assembled genome reconstruction from environmental samples, enabling a detailed phylogenomic and metabolic analysis. The genome of umkhey_bin13 (proposed as ‘Candidatus Viridifilum namsaraevi’ gen. nov., sp. nov.) encodes a complete 3-hydroxypropionate bicycle for autotrophic CO₂ fixation, a type II sulfide:quinone oxidoreductase, but lacks nitrogenase and chemolithotrophic markers. Comparative genomics across 18 Chloroflexota genomes reveals that this new lineage occupies an intermediate phylogenetic position, with distinct gene content for sulfur, nitrogen, and hydrogen metabolism, and highlights a complex evolutionary history within the group. Our findings provide new insights into the evolutionary transitions of photosynthetic and carbon-fixation pathways, and underscore the value of integrating cultivation-independent genomics with classical microbiology. The proposed novel genus and species contribute to a revised framework of Chloroflexota evolution and offer a genomic reference for studying metabolic adaptation in alkaline, sulfide-influenced microbial mats.

In Germany, maize (Zea mays L.) is cultivated on approximately 2.4 Mha, making it one of the most widely grown arable crops alongside wheat and barley (German Federal Statistical Office, 2025). Although maize has previously been identified as a host of Candidatus Phytoplasma solani (PHYPSO, 16SrXII-A), particularly in relation to maize redness (MR) disease detected in southeastern Europe (Jović et al., 2009), its role as a host of ‘Candidatus Arsenophonus phytopathogenicus’ (ARSEPH) has not been reported previously. The planthopper Pentastiridius leporinus (Hemiptera: Cixiidae) is the major vector for transmitting both pathogens in many crops in Germany (Rinklef et al., 2024). MR symptoms include patchy premature drying, reddish discoloration of leaves and midribs, poorly filled or aborted ears, and malformed kernels. In August and September 2025, maize plants showing MR-like symptoms were observed in different fields in Otzberg 49°48'25.3"N, 8°52'28.8"E (South Hesse), Bellheim 49°10′19.9N, 8°15′50.8E, Bockenheim 49°36'18.6"N, 8°11'30. E"O, Ibersheim 49°43′36.5″N, 8°24′34.6″E (Rhineland-Palatinate) and Ettling 48°48'50.0"N, 11°40'17.2"E (Baden-Württemberg). Samples from roots (25), leaves (10), ears (14) and kernels (9) of 14 selected symptomatic plants were collected for molecular analysis. DNA was extracted using a CTAB-based protocol. ARSEPH was detected by qPCR targeting the manA gene according to Zübert and Kube (2021). Positive results were obtained from ears of six plants from Ibersheim and Ettling, leaves of three plants from Bockenheim and Otzberg, kernels of two plants from Bellheim, and roots of one plant from Bockenheim. Four ARSEPH-positive samples (two ears and two kernels) were further analyzed using the Fra4/Fra5 PCR assay (Zreik et al. 1998). Sanger sequencing of the resulting amplicons followed by BLAST analysis confirmed all four sequences as ‘Candidatus Arsenophonus phytopathogenicus’ with 100% identity to reference sequence PP400342. The sequences have been deposited in GenBank under accession numbers PZ623476, PZ623478, PZ623483, and PZ626926. Furthermore, all samples were screened for PHYPSO using the qPCR assay described by Behrmann et al. (2022) with primers KL464/KL465. Seven ear samples from Ibersheim yielded positive qPCR signals. To further validate these results, DNA from qPCR-positive samples was subjected to nested PCR targeting the 16S rRNA gene using primer pairs P1/P7 (Schneider et al., 1995) and R16F2n/R16R2 (Gundersen and Lee, 1996), followed by Sanger sequencing and BLAST analysis against the NCBI GenBank database. However, repeated sequencing did not provide sufficient sequence identity to the reference strain STOL11 (GenBank accession number AF248959) to confirm the qPCR-positive samples as PHYPSO. To our knowledge, this is the first report of ARSEPH in maize in Germany. Its detection in maize extends the known host range of this phloem-limited bacterium. The high ARSEPH loads in ears and kernels detected in reproductive tissues are consistent with preferential accumulation in sink organs during grain filling. Whether maize contributes to pathogen persistence and transmission within the Pentastiridius leporinus or other potential vectors’ pathosystem remains to be investigated.

Resumen. Interpolación espacial de Candidatus Liberibacter asiaticus1 junto Con su vector Diaphorina citri2 en Tamaulipas, México: Un enfoque geoestadístico incorporando variables climáticas La producción de cítricos en Tamaulipas, México, se ha visto afectada por el Huanglongbing (HLB), una enfermedad de distribución global en árboles cítricos causada por la bacteria Candidatus Liberibacter asiaticus Jagoueix (CLas) (Hyphomicrobiales: Rhizobiaceae). Debido a la falta de tratamiento para el patógeno, los métodos de control se han centrado principalmente en su vector: el psílido asiático de los cítricos, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), para combatirlo. Para apoyar y mejorar la vigilancia y el control de la propagación del HLB, este estudio empleó métodos geoestadísticos para modelar y predecir la distribución espacial continua de CLas. Empleamos Kriging Ordinario (KO) y CoKriging Ordinario (CKO) empleando covariables climáticas, temperatura media y máxima, precipitación y número de días lluviosos. La validación cruzada mostró que el modelo CKO que incorporo la precipitación (CKO -P) tuvo el mejor desempeño predictivo, seguidos por los que incluían temperatura media (CKO -Tmed) y máxima (CKO -Tmax), mientras que KO y CKO con días lluviosos (CKO -D) presentaron un rendimiento menos eficiente. Los mapas de interpolación identificaron a Güémez, Victoria y Padilla como los principales puntos críticos, coincidiendo con los grupos previamente reportados. Tula, Jiménez, Altamira, Abasolo y Soto la Marina fueron identificados como áreas climáticamente favorables fuera de la región tradicional de producción de cítricos y más allá de los grupos significativos. Estos resultados indican que la predicción de la ocurrencia de CLas y D. citri no depende solo de la proximidad a casos previamente registrados, sino que también puede mejorarse con la variabilidad ambiental. Esto amplía la información de las zonas de vigilancia, lo que puede fortalecer el monitoreo epidemiológico y el manejo regional del HLB en Tamaulipas y otras regiones.