Publications

4877

ABSTRACT Acidophilic ammonia-oxidizing bacteria (AOB) have only recently been discovered. These organisms hold great promise for acidic wastewater treatment; however, their physiology remains poorly understood compared to that of neutrophilic AOB. Here, we investigated the physiology of the acidophilic AOB “Candidatus Nitrosacidococcus tergens ” sp. RJ19 across a broad pH range (2.5–7.0) using a specialized bioreactor system. We monitored nitrogen (N) transformations combined with microbial community composition and transcriptomic profiles, focusing on nitrogen metabolism and proton stress responses. Our results show that above pH 6.0, “ Ca . Na. tergens ” performs complete and stoichiometric conversion of ammonium to nitrite, coinciding with isotopic fractionation effects specific for ammonia oxidation and increased expression of key ammonia oxidation genes. The apparent absence of nirK and cycA did not impede ammonia oxidation, suggesting that these genes are non-essential in this context. Below pH 6.0, nitric oxide and nitrate accumulated, and nitrous oxide (N₂O) levels, although negligible compared to the other N-compounds, peaked near pH 4.0. Stable isotope analysis, including the site-specific 15 N-enrichment at the inner (α) and outer (β) nitrogen positions of the N 2 O molecule, indicated nitrifier-denitrification as the source of N₂O, supported by the highest norB expression at this pH. These findings provide new insights into the acid-tolerant physiology of “ Ca . Na. tergens ” and advance its potential application in engineered nitrogen removal systems under acidic conditions. IMPORTANCE The world is facing a climate crisis intensified by human-driven nutrient pollution. Ammonia and the bacteria that oxidize it are central both to the global nitrogen cycle and to wastewater treatment. The acidophilic ammonia oxidizer “ Candidatus Nitrosacidococcus tergens” was previously shown to oxidize ammonia under highly acidic conditions; however, a complete understanding of its metabolism is lacking. Our study now shows that “ Ca . Na. tergens” performs canonical ammonia oxidation across a broad pH range. At pH values below 6, however, a combination of chemical and biological processes leads to the production of nitrate, nitric oxide, and the greenhouse gas nitrous oxide. In addition, we show that these bacteria adapt to proton stress through mechanisms beyond transcriptional mechanisms. Our study highlights the robust metabolism of acidophilic ammonia oxidizers and expands our understanding of nitrification under acidic conditions.

Abstract ‘ Candidatus Liberibacter solanacearum’ (Lso) is a devastating bacterial pathogen of crops transmitted by psyllids. Previously, a link between changes in ubiquitination and the progression of diseases caused by Lso was established. Among the Lso haplotypes, LsoA and LsoB infect solanaceous crops. LsoA induces chlorosis and stunting in tomato, while LsoB infection leads to more severe symptoms, including plant death. To better characterize the molecular basis of the differences in the diseases caused by these haplotypes, we cataloged the ubiquitinome of tomato leaves four weeks after LsoA and LsoB-infection and following infestation with Lso-free psyllids. Our analysis revealed haplotype-specific changes in the plant ubiquitinome. Gene Ontology (GO) terms related to glucose metabolism and proteasome-mediated ubiquitin-dependent protein catabolism were enriched among the differentially ubiquitinated proteins between LsoA-infected and Lso-free infested plants. Additionally, a KEGG pathway analysis determined an overrepresentation of proteins involved in carbon metabolism and fixation via the Calvin cycle. In contrast, the comparison of LsoB-infected and Lso-free infested plants revealed a broader range of enriched GO terms and KEGG pathways, including those associated with carbohydrate and energy metabolism, proteasome activity, and oxidative stress. These results contribute to our understanding of the development of Liberibacter-related diseases and reveal the importance of ubiquitin-related pathways in Lso infection. They also contribute to the expanding inventory of ubiquitinated proteins.

The bois noir (BN) disease of grapevines is widespread in German winegrowing regions. It is associated with ‘Candidatus Phytoplasma solani’, which affects not only grapevines but also other wild and cultivated plants. This pathogen has a complex epidemiology including different insect vectors and various host plants. A study was carried out to investigate the genetic variability of ‘Ca. P. solani’ in different winegrowing regions in Germany. Between 2017 and 2023, samples of grapevine, stinging nettle, bindweed, and other herbaceous plants as well as specimens of different planthopper species colonizing viticultural habitats were analyzed for infection with ‘Ca. P. solani’. All positive tested samples were further characterized by multilocus sequence typing (MLST) based on the genes tuf, stamp, secY, and vmp1. The genetic variability was assessed by RFLP analyses of the tuf and vmp1 PCR products, coupled with sequencing of the stamp and secY amplification products. A total of 1274 grapevines, 35 bindweed, and 18 stinging nettle samples were infected with ‘Ca. P. solani’ but also five samples of other weed species. Among the known and putative insect vectors, specimens of Hyalesthes obsoletus, Reptalus spp., and Dictyophara europaea harbored the phytoplasma. In both plants and insects, two genotype combinations were predominantly associated with the classical bindweed and stinging nettle cycle, respectively. The MLST analysis revealed considerable differences between German isolates and data reported from other European regions and new genotype combinations were identified, indicating new host plant–vector associations.

‘Candidatus Liberibacter asiaticus’ (CLas) is a devastating pathogen of citrus associated with Huanglongbing (HLB, citrus greening disease). HLB is economically significant in Asia and has destroyed millions of citrus trees worldwide during the last century. Since 2007, when the first molecular evidence for the presence of CLas in North-West Frontier Province of Pakistan (Khyber Pakhtunkhwa) were presented, there have been a limited number of studies reporting the pathogen in different citrus growing districts of the Punjab Province, one of the major citrus producing areas of the country. In this study, a small-scale survey was conducted in citrus groves exhibiting HLB-like symptoms in different districts of Punjab Province. The aim was to obtain current information on the incidence of CLas in the area and complement the previous studies. Conventional and real time quantitative polymerase chain reaction (PCR and qPCR) assays were used for the detection of CLas in the 94 samples tested, followed by sequencing of the PCR amplicons. Overall CLas was detected in 43% (40/94) of the tested samples from Kinnow mandarin (n= 22), sweet orange (n= 12), grapefruit (n= 3) and Feutrell’s early mandarin (n= 3). In agreement with previous reports, this survey confirmed the presence of HLB in the Punjab Province of Pakistan and provided updated molecular evidence for the presence of CLas and disease incidence for four citrus cultivars from five citrus growing districts of the province.

ABSTRACT Ubiquitin‐proteasome is a conserved mechanism that regulates cellular responses and disease resistance in plants. However, the regulatory role of ubiquitin‐proteasome in the pathogenicity of “ Candidatus Liberibacter asiaticus” ( C Las), the causal agent of citrus huanglongbing (HLB), one of the most serious citrus diseases, remains poorly defined. In this study, we demonstrated that CLIBASIA_04250 (SDE4250) suppresses 26S proteasome activity in citrus, leading to a reduction in salicylic acid (SA) levels and downregulation of SA‐responsive genes. Further investigation revealed that SDE4250 specifically targets the citrus 26S proteasome non‐ATPase regulatory subunit 4 homologue (CsRPN10) and enhances its degradation via the 26S proteasome pathway. Moreover, overexpression of CsRPN10 inhibited C Las proliferation in citrus hairy roots, which was accompanied by a significant accumulation of SA content and upregulation of related defence genes. Collectively, our findings indicate that SDE4250 targets the citrus 26S proteasome to disrupt SA‐mediated citrus immunity.

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.