Publications

4495

Abstract Background ‘ Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited bacterial pathogen causing significant diseases in solanaceous crops. In the United States, haplotypes A and B are transmitted by the potato psyllid Bactericera cockerelli . We previously identified differences in their acquisition and transmission between adults and nymphs. The present study characterized the dynamics of LsoA and LsoB acquisition and transmission by nymphs and examined the transcriptional responses of the nymphal gut upon their acquisition. Methods and results Nymphs were exposed to LsoA- or LsoB-infected plants for 1, 3, 5, or 7 days to measure the bacterial accumulation and for 8 days to assess the transmission efficiency following sequential inoculation of tomato plants. Quantitative PCR showed that LsoB accumulated to higher levels than LsoA after 3 days of acquisition. Following the sequential inoculation, LsoB was transmitted earlier than LsoA indicating a shorter latency period. RNA-seq analysis of the guts following a 1- and 5-day acquisition access periods revealed a greater transcriptional regulation at 5 days than at 1 day. Furthermore, the responses were haplotype-specific: LsoA primarily affected genes involved in protein translation, ER stress, and cell cycle regulation, whereas LsoB regulated genes involved in autophagy, apoptosis, and immune pathways. Conclusions This study revealed haplotype-specific gene regulation potentially leading to LsoB being transmitted more efficiently by psyllid nymphs.

ABSTRACT Citrus Huanglongbing (HLB), caused by ‘ Candidatus Liberibacter asiaticus’ ( Ca Las), is the most devastating disease affecting the global citrus industry. Here, we reported that the Ca Las effector SDE70 promotes HLB pathogenicity by targeting the citrus ubiquitination pathway. Transgenic expression of SDE70 in Wanjincheng orange ( Citrus sinensis Osbeck) accelerated early Ca Las proliferation, aggravated HLB symptoms, and increased susceptibility to citrus canker induced by Xanthomonas citri subsp. citri ( Xcc ). These results demonstrate that SDE70 functions as a broad‐spectrum suppressor of citrus immunity. Mechanistically, SDE70 physically interacts with CsRUB2, a citrus ubiquitin‐related protein. Furthermore, CsRUB2 overexpression in Wanjincheng oranges reduced resistance to HLB but enhanced resistance to citrus canker. Both SDE70 and CsRUB2 elevated salicylic acid (SA) and hydrogen peroxide (H 2 O 2 ) levels in transgenic plants while lowering methyl salicylate (MeSA) levels. CsRUB2 also decreased jasmonic acid (JA). In contrast to the suppressive effect of SDE70, CsRUB2 enhanced the transcription of citrus immunity genes. Transient expression assays further demonstrated that the SDE70–CsRUB2 interaction dysregulates citrus immunity by perturbing SA, MeSA, JA, and H 2 O 2 signals. These findings provide a theoretical basis for understanding citrus– Ca Las interactions and breeding citrus varieties with broad‐spectrum resistance to both HLB and citrus canker.

Abstract ‘Candidatus Mycoplasma haemolamae’ (CMhl) is a hemotropic bacterium infecting South American Camelids (SAC), whose epidemiology and clinical significance are still not fully elucidated. This study investigated CMhl by qPCR in blood samples of alpacas ( Vicugna pacos ) and llamas ( Lama glama ) in Italy in 2021–2024, characterizing CMhl haplotypes based on partial 16 S rRNA sequences and defining haematological findings in a subset of CMhl-infected animals. Statistical analysis was performed to detect potential risk factors associated with CMhl positivity. Out of 206 animals (200 alpacas and 6 llamas), CMhl was detected by qPCR in 88 (42.7%) animals. CMhl haplotype #1 and #2 were detected in 42.2% and 0.5% of the animals, respectively. Binary logistic regression analysis showed that higher CMhl positivity was significantly associated with larger herd size ( P  = 0.011), Northern Italy area of origin ( P  = 0.031) and warmer seasons ( P  = 0.003). The detection of two CMhl haplotypes confirms the high diffusion of CMhl in SAC farmed in Italy. Results confirm that CMhl usually causes subclinical infections, suggesting that antimicrobial treatment is likely not necessary based on qPCR positivity alone. Results focusing on the association of CMhl positivity with warm temperatures and large herd size suggest the need of further studies on the genetic features, epidemiology and immune response in CMhl-positive SAC from Italy, with emphasis on understanding potential vector transmission.

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.

‘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.

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.

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 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.