Publications

4719

Citrus huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (CLas), is a destructive disease threatening global citrus industry. Although citrus cultivars differ in HLB sensitivity, how infection alters endophytic bacterial communities in cultivars with contrasting susceptibility remains unclear. Here, we compared endophytic microbiome shifts in leaf and root tissues of HLB-susceptible Shatangju mandarin (Citrus reticulata cv. Shatangju) and HLB-tolerant Shatian pomelo (C. maxima cv. Shatian) at 10 months postgrafting with CLas-infected buds. Infected Shatangju mandarin showed severe symptoms and higher CLas levels in leaves than in roots, while infected Shatian pomelo remained asymptomatic with higher CLas in roots than in leaves. High-throughput 16S rRNA gene sequencing revealed that CLas infection restructured the endophytic microbial community in a tissue-specific manner. CLas-infected Shatian pomelo leaves displayed increased microbial diversity and network complexity, while CLas-infected Shatangju mandarin roots harbored more disease-suppressive taxa like Streptomyces. Across both cultivars, network complexity correlated inversely with CLas abundance. Key genera (e.g., Pseudomonas, Streptomyces, and Prevotella) correlated positively with CLas, suggesting roles in mitigating pathogen effects. Predicted functional profiling indicated activated pathways (amino acid metabolism, terpenoid/polyketide biosynthesis, and xenobiotic biodegradation) potentially supporting host defense in infected tissues. The HLB tolerance of Shatian pomelo appeared linked to tissue-specific bacterial restructuring, particularly recruitment of antibiotic-producing bacteria and enhanced metabolic resilience. These findings elucidated cultivar-specific microbial strategies against CLas, offering insights for microbiome-mediated HLB management.

ABSTRACT Insect‐vectored pathogens pose a significant threat to global agriculture. The colonization efficiency of pathogens in vectors plays a central role in these pathosystems, yet studies of the factors that affect this aspect are limited. This study investigates the genetic and microbial symbiont factors influencing the susceptibility of Diaphorina citri to Candidatus Liberibacter asiaticus ( C Las), the pathogen causing citrus greening disease (huanglongbing). Through a microbiome Genome Wide Association Study (mGWAS) based on 16S amplicon sequencing and genomic resequencing of 120 D. citri individuals from six populations, we identified 79 SNPs significantly associated with the relative abundance of C Las within insects. Additionally, some of these SNPs were also associated with the relative abundance of Candidatus Profftella armature, a key endosymbiont of D. citri . SNPs in the regulatory region of gene Dcitr04g11610.1 led to its overexpression in C Las‐susceptible D. citri , and C Las infection further elevated its expression. Conversely, RNAi knockdown of Dcitr04g11610.1 reduced C Las infection rates and abundance, accompanied by increased abundance of Profftella . Phylogenetic analysis revealed Dcitr04g11610.1 ’s high homology to Major Facilitator Superfamily‐type transporter SLC18B1 proteins, suggesting a role in C Las polyamine utilization. These findings highlight the importance and potential interplay of insect genetics and symbiotic microbiota in insect‐vectored plant pathogen systems.

'Candidatus Phytoplasma solani' is a distinct species of the class Mollicutes (16SrXII group), characterized by phloem-limited localization and strong host dependence. It is widely distributed, mainly in Europe, and affects over 100 plant species, including economically important crops for Moldova. The name reflects its significant impact on plants of the Solanaceae family, causing stolbur disease. It also infects grapevine, pepper, maize, lavender, and other crops. Climate change has favored the spread of the pathogen and its insect vectors into new regions and host plants. Therefore, monitoring and controlling its dissemination in economically important crops is essential. Accurate diagnosis can be achieved by molecular methods, with PCR-based techniques offering a reliable and cost-effective approach. The aim of the study was to determine the presence or absence of infection in some agricultural crops: tomato, maize, potato, pepper, eggplant, wheat, plum and apple growing in experimental fields of Institute of Genetics, Physiology and Plant Protection, MSU. Materials and methods: Various DNA extraction methods were used depending on plant culture: boiling in alkaline solution, DNAzol, SDS, CTAB. Nested PCR analysis was carried out using specific to 'Ca. P. solani' primers designed on the base of chaperonine or 16S rRNA gene sequences. Amplification results were recorded using gel agarose electrophoresis (1.5% agarose, 0.5 x TBE buffer) stained with ethidium bromide and visualized in UV. Results: A comprehensive study of tomato conducted during 2016-2025 showed the continuous presence of the pathogen, with infection rates reaching up to 72% depending on annual conditions, confirming the high susceptibility of tomato to 'Ca. P. solani'. In potato, 14.6% of plants were infected, while sweet pepper showed a much lower incidence of 4%. Overall, the results indicate the predominant distribution of the pathogen within the Solanaceae family, further supported by its detection in the ruderal species Solanum nigrum. The phytoplasma was not detected only in eggplant among the analyzed Solanaceae species. In contrast, molecular diagnostics gave negative results in crops from other families, including maize and wheat (cereals), as well as apple and plum (fruit crops). The pathogen was not detected in orchard trees during either the dormant or fruit-bearing stages. Conclusions: Thus, obtained results demonstrate an ecological and epidemiological specificity of 'Ca. P. solani' for certain host species, characterized by a high incidence in Solanaceae, particularly tomato. Additionally, the study confirms the potential role of ruderal species within the same family as natural reservoirs of the pathogen. These data contribute to a deeper understanding of the spread of the phytoplasma providing a base for effective monitoring and phytosanitary management strategies.

Abstract Magnetotactic bacteria form a highly diverse group of microorganisms, yet early exploration of their diversity was largely centered on the Pseudomonadota. More recently, metagenomic studies have revealed that magnetotaxis, a form of chemotaxis guided by Earth's magnetic field, is widespread in other deep-branching phyla for which little to no ecological or biological information is available beyond that inferred from their genomes. For most of them, the morphology, ultrastructure and magnetosome chain characteristics responsible for the magnetic guidance remain unknown. While screening extreme environments for novel magnetotactic species, we observed magnetotactic Bdellovibrionota in the anoxic and ferruginous sediments of the Fontaine Goyon spring (France). We characterized their cell morphology and ultrastructure using magnetic enrichment, a single-cell sorting approach, and high-resolution electron microscopy. Cells display the morphology typical of the few predatory bacteria described in this phylum, and biomineralize, on average, five irregularly faceted, bullet-shaped magnetite magnetosomes along the concave side of the cell. Metagenomic analysis of approximately one hundred cells revealed a potentially predatory and heterotrophic lifestyle adapted to low-O2 conditions. It also suggests a flexible respiratory metabolism under varying redox conditions, using iron as an alternative terminal electron acceptor. Exploring the diversity of Bdellovibrionota in public databases, we found 21 metagenome-assembled-genomes containing magnetosome genes. None of them harbor the canonical mamK actin-like gene implicated in aligning magnetosomes in described magnetotactic models. Affiliated to an undescribed class, we propose a classification scheme for the magnetotactic Bdellovibrionota species representing the class Bdellonasia class nov., for which no species had been formally described.

ABSTRACT The oil refinery (OR) consortium is a model methanogenic enrichment culture used to study anaerobic benzene degradation. Over half of the culture’s bacterial community consists of two closely related Desulfobacterota strains, designated ORM2a and ORM2b, whose mechanisms of benzene activation are unknown. Three proteomics data sets were integrated and analyzed using high-quality OR metagenomes and metagenome-assembled genomes (MAGs), including a complete circularized ORM2a MAG, to identify active metabolic pathways and proteins expressed during methanogenic benzene degradation. Among the proteins identified were Bam-like subunits of an ATP-independent benzoyl-CoA degradation pathway, as well as downstream β-oxidation proteins yielding acetate. The most abundant proteins identified mapped to two ORM2a gene clusters of unknown function. Homologous and syntenic gene clusters were identified in the MAGs of ORM2b and a sulfate-reducing Pelotomaculum that also degrades benzene, as well as in nine contigs assembled from hydrothermal vent metagenomes. Extensive homology and structural predictions suggest that the first cluster—termed the “Magic” gene cluster—encodes for enzymes catalyzing the chemically challenging activation of benzene and subsequent transformation steps yielding benzoyl-CoA. The second (“Nanopod”) gene cluster encodes a transmembrane complex that may facilitate benzene transport across the cell membrane. Phylogenomic analyses place ORM2a and ORM2b within a novel genus of strict anaerobes specialized for benzene degradation, which we propose naming “ Candidatus Anaerobenzenivorax.” IMPORTANCE Benzene is a widespread, persistent, and toxic pollutant that can accumulate in anoxic environments such as groundwater and sediments. Benzene can be metabolized in the absence of oxygen; however, despite decades of research, the biochemical mechanisms for benzene activation under anaerobic conditions remain unproven. This study provides strong genetic and proteomic evidence for a new suite of enzymes that initiate anaerobic benzene activation. These findings lay a foundation for future biochemical studies and expand our understanding of how microbes carry out difficult chemical reactions in the absence of oxygen.

A hydrogenotrophic methanogen, designated strain ACI-7 T , was isolated from an alkaline, saline wetland located in eastern Nebraska, USA. Cells were identified as non-motile rods (0.9–4.2 µm in length and 0.2–0.4 µm in diameter) that occurred singly, in chains, or as twisted chains and aggregates. Strain ACI-7 T utilized H 2 +CO 2 or formate as methanogenic substrates and required growth factors present in yeast extract for continuous cultivation. The strain grew at 20–45 °C (optimum, 40 °C), at pH 6.5–8.5 (optimum, pH 7.3) and with 0–2.5% NaCl (optimum, 0–1%). The genomic G+C content of ACI-7 T was 31.79 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated strain ACI-7 T was affiliated with the genus Methanobacterium , most closely related to Methanobacterium oryzae FPi T (97.1% sequence similarity) and Methanobacterium veterum MK4 T (96.9% sequence similarity). Overall genome relatedness indices for strain ACI-7 T compared to other Methanobacterium species ranged from 68.45 to 78.17% for average nucleotide identity and 18.9 to 25.7% for digital DNA–DNA hybridization. Morphological, physiological and genomic characteristics indicate that strain ACI-7 T represents a novel species, for which the name Methanobacterium nebraskense sp. nov. is proposed. The type strain is ACI-7 T (=DSM 118696 T =ATCC TSD-487 T ).

‘Candidatus Phytoplasma rubi’ (elm yellows group, 16SrV-E phytoplasma subgroup) is the causal agent of rubus stunt disease, a disorder affecting economically important plants—raspberries and blackberries. Although this phytoplasma has been extensively studied in cultivated Rubus crops, its occurrence and molecular identity in wild Rubus species populations in North-Eastern Europe remain poorly documented. In this study, phytoplasmas associated with symptomatic wild Rubus idaeus L. and Rubus nessensis Hall plants were investigated in natural forest ecosystems of the Curonian Spit, Lithuania. A total of 65 symptomatic plants were surveyed, and phytoplasma infection was detected in 30 samples by nested PCR targeting the 16S rRNA gene. Positive samples were characterized using a multilocus molecular approach based on sequence analysis of the additional cpn60 and secA genes. All strains showed high nucleotide sequence similarity across the analysed loci and consistently clustered with reference strains of ‘Candidatus Phytoplasma rubi’. Virtual RFLP profiles derived from the 16S rRNA and cpn60 genes were nearly identical to those of established 16SrV-E phytoplasma subgroup reference strains and clearly distinct from other 16SrV phytoplasma subgroups. These results provide not only the first detailed multilocus molecular characterization of ‘Candidatus Phytoplasma rubi’-related strains infecting wild Rubus species in Lithuania but also represent the first report of this phytoplasma in naturally occurring R. idaeus and R. nessensis plants in the country, thereby extending the known geographical occurrence of this pathogen and documenting its presence in wild Rubus hosts from unmanaged forest habitats in the Eastern Baltic region of Northern Europe.

Ocean warming, disease, and pollution have contributed to global declines in coral abundances and diversity. In the Caribbean, corals previously dominated reefs, providing an architectural framework for diverse ecological habitats, but have significantly declined due to infectious disease and anthropogenic climate change. Key species like the coral Acropora cervicornis are critically endangered, prompting researchers to focus on scientific endeavors to identify factors influencing coral disease resistance and resilience. We previously showed that disease susceptibility, growth rates, and bleaching risk were all associated with the abundance of a single bacterial parasite, Candidatus Aquirickettsia rohweri which proliferates in vivo under nutrient enrichment. Yet how nutrients influence parasite physiology in vivo remains unknown. Here, we analyzed parasite gene expression from a disease-susceptible A. cervicornis genotype exposed to ambient or nutrient enrichment conditions. Electron microscopy showed that Ca. A. rohweri was abundant in coral tissue and densely packed in mucocytes prior to nutrient enrichment. Under ambient conditions, the parasite upregulated genes involved in translation, protein maintenance, and cell envelope integrity, consistent with a conserve-and-maintain strategy. Nutrient enrichment induced expression of genes associated with central metabolism, nutrient import, stress response, host interaction, and two-component systems. Together, these results indicate that nutrient enrichment activates a growth-and-exploitation strategy, likely exacerbating parasitic pressure on A. cervicornis .

A field investigation was conducted during the winter seasons of 2021–22 and 2022–23 at the Jawaharlal Nehru Krishi Vishwa Vidyalaya (JNKVV), Jabalpur, Central India, to identify and characterize the phytoplasma strains associated with niger (var. JNC6) and sesame (var. RT315) exhibiting severe phyllody symptoms. Disease incidence ranged from 0-3%, and 3 to 15% were recorded in niger and sesame crops during the study period. Nested PCR assays using universal phytoplasma primer pairs P1/P7 followed by R16F2n/R16R2 consistently amplified ~1.25 kb fragments of the 16S rRNA gene from both the symptomatic plants, confirming phytoplasma association. Additional amplification of phytoplasma-specific sec A (~600 bp) and sec Y (~1.7 kb) gene fragments further validated the phytoplasma infection in both crops. Two predominant leafhopper species, Amrasca biguttula and Orosius albicinctus , colonizing and feeding niger and sesame fields were identified. Notably, only Amrasca biguttula was tested positive for phytoplasma using the similar PCR assays mentioned above, implicating it as a putative vector. Sequence identity, phylogenetic analysis, and in silico RFLP profiling of 16S rRNA, sec A, and sec Y gene sequences from both the symptomatic plants and leafhoppers confirmed the presence of a ‘ Ca. P. asiaticum’ related strain (16SrII-C subgroup). This study represents the first global report of 16SrII-C subgroup phytoplasma infection in niger, highlighting a new host record. The detection of an identical phytoplasma strain in niger, sesame, and Amrasca biguttula suggests a possible epidemiological link and underscores the role of these natural hosts and the leafhopper in the dissemination of ‘ Ca. P. asiaticum’-related phytoplasma strains in Central India.