Publications

4721

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.

Peptidyl-tRNA hydrolase (Pth2) hydrolyzes peptidyl-tRNA, an immature product of aborted translation, into peptide and tRNA, thereby maintaining cellular protein synthesis through peptide release and tRNA recycling. Here, we present the crystal structure of Pth2 from Candidatus Lokiarchaeum sp. GC14_75 ( Loki Pth2) at 2.12 Å resolution. This is the first structure of Pth2 from a lineage within Promethearchaeati, a kingdom of archaea closely related to eukaryotes. The structure reveals that Loki Pth2 forms a homodimer and closely resembles Pth2 structures from other species. However, Loki Pth2 exhibits two prominent structural differences: a short helix around the catalytic center, which is absent in other Pth2s, and a distinct orientation of the C-terminal helix. Detailed comparative structural analysis suggests that these regions may regulate enzymatic activity and substrate binding, respectively. Furthermore, the corresponding regions in other Pth2s also exhibit high flexibility, suggesting that similar mechanisms may be conserved among Pth2s. To gain insights into the growth environment of Candidatus Lokiarchaeum sp. GC14_75, we assess the optimal temperature for the catalytic reaction of Loki Pth2, which suggests that Candidatus Lokiarchaeum sp. GC14_75 inhabits moderately thermophilic environments.

Introduction Huanglongbing (HLB) is a devastating disease impacting citrus orchards worldwide. Identifying candidate genes and/or genomic regions that significantly influence plants' response to HLB is essential for breeding programs. Nevertheless, this remains a challenge, especially during the early stages of the citrus-pathogen interaction, when phenotypically distinguishing tolerant genotypes is not trivial. This work focused on identifying genomic regions and candidate genes associated with citrus response to Candidatus Liberibacter asiaticus ( C Las). Methods Quantitative trait loci (QTL) mapping was performed in a citrandarin population (278 individuals) derived from a fullsib cross between susceptible Citrus sunki (CS) and tolerant Poncirus trifoliata (PT). The mapping population was genotyped using DArTseq markers and the SNPs obtained from them (DArTseq-derived SNPs), as well as the EMBRAPA Multispecies 65K Illumina Infinium® chip. Phenotyping was based on four individual trials (two in a greenhouse and two in field conditions). QTL mapping was based on composite interval mapping implemented in the fullsibQTL software, with the cycle threshold (Ct) of quantitative PCR (qPCR) as the trait of interest for assessing C Las quantification in infected tissues, which can be evaluated in plants at any developmental stage. The biological function of QTLs was investigated by gene annotation using the chromosome-level phased Citrus sinensis genome (assembly DVS_A1.0) as the reference. The candidate genes were functionally annotated using Blast2Go software. Results A predominance of markers in a 1:1 ratio was observed, resulting in two maps, one for each parent. The linkage map had 5464 cM (1872 markers) for CS and 4794 cM (1340 markers) for PT. A total of 44 QTLs were mapped: 25 for the CS map (13 for field and 12 for greenhouse conditions), of which 17 were located at gene sites; and 19 were mapped for the PT (nine for field and 10 for greenhouse conditions), of which 16 were located at gene sites. Discussion Several genes associated with the identified QTLs are involved in stress responses, making them potential targets for further functional validation. These genomic regions and candidate genes provide valuable targets for future incorporation into citrus breeding programs aimed at developing HLB tolerance.

Little ironweed (Cyanthillium cinereum; syn. Vernonia cinerea) is an herbaceous plant in the Asteraceae family (Trang et al. 2024). The plant is distributed in Asia, Australia and Africa, and has been used as a traditional medicine (Trang et al. 2024). In January 2026, little ironweed plants exhibiting phyllody symptoms (Fig. S1) were observed in a grass-covered area (measuring 3 m × 6 m) in Qishan District, Kaohsiung, Taiwan. Around 8% (22/285 plants) of the population showed symptoms. Six symptomatic and four asymptomatic plants were collected. Flower-like tissues from symptomatic plants were collected for DNA extraction with a Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics). For asymptomatic plants, capitula were used instead. One DNA sample was extracted for each plant. The samples were first tested with primers specific to the plant’s 26S rDNA (28KJ/28C; Cullings 1992), and all of them produced the target amplicon (~0.69 kb), indicating sufficient quality for polymerase chain reaction (PCR). Nested PCR targeting phytoplasmas’ 16S rDNA was conducted with outer and inner primer pairs P1/P7 and fU5/rU3 (Lorenz et al. 1995). In all PCR assays, negative controls (nuclease-free water) were included. All samples from symptomatic plants amplified the outer and inner amplicons (~1.81 kb and ~0.88 kb), whereas DNA from the asymptomatic plants did not. After assembling sequences of the outer and inner fragments for each symptomatic sample, identical 16S rDNA sequences were found. A representative sequence was deposited in GenBank (1,459 bp; accession no. PZ193724), and BLASTn search against GenBank revealed that it was identical to that of a reference phytoplasma strain belonging to subgroup 16SrII-A (NCHU2014; accession no. CP040925; 100% identity, 100% query coverage; Rodrigues Jardim et al. 2023). A 1,248-bp fragment within the obtained sequence was further analyzed using iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi), and its virtual RFLP pattern was the same as 16SrII-A’s reference pattern (similarity coefficient = 1.00; accession no. L33765). The detected phytoplasma’s identity was further confirmed by amplification and sequencing of the elongation factor Tu (tuf) and protein translocase (secY) genes. Fragments containing tuf and secY were respectively amplified by primer sets TUF-II-F1/TUF-II-F2/TUF-II-R1 (for semi-nested PCR; Al-Subhi et al. 2017) and SecYF1(II)/SecYR1(II) (for PCR; Lee et al. 2010). The tuf sequences were obtained from the inner amplicon, whereas sequences of secY were obtained by sequencing followed by primer walking (accession nos. PZ377067 and PZ377068). The detected phytoplasma and the reference strain NCHU2014 (‘Ca. Phytoplasma australasiaticum’-related strain; 16SrII-A; accession no. CP040925) shared identical sequences for both tuf (986 bp; partial) and secY (1,263 bp; complete) fragments. This is the first report of little ironweed infected by a 16SrII-A phytoplasma in Taiwan; this specific host-pathogen association has only been reported in Hainan Province of China (Yu et al. 2024). Because 16SrII-A phytoplasma can infect economic crops in Taiwan (Wu et al. 2026) and little ironweed is widely distributed in the island, it is possible that the weed may represent a potential reservoir host. Thus, monitoring and eradication of symptomatic little ironweed is advisable in crop production areas.

ABSTRACT We present the complete de novo assembly of a Candidatus Walczuchella monophlebidarum genome (286,606 bp), a flavobacterial endosymbiont of the giant-scale insect Icerya purchasi . The genome was assembled from metagenomic short read Illumina sequences obtained from DNA of citrus leaves collected in Carinthia, Austria in November 2024.

Pear decline, associated to ‘Candidatus Phytoplasma pyri’, has emerged in Chilean pear orchards in recent years. While several Cacopsylla species are potential vectors of ‘Ca. P. pyri’, the disease’s full epidemiological cycle remains uncertain. Cacopsylla bidens, present in Chilean orchards, has recently been reported as a vector. This study conducted year-long surveys in two commercial pear orchards across different Chilean regions, capturing C. bidens in zones with ‘Ca. P. pyri’-infected pear trees. All developmental stages were collected, with peak abundances occurring in March and April. Outside the study zones, C. bidens were found in pine trees but not in adjacent cultivated areas. Two seasonal morphotypes, summer and winter forms, were identified. Molecular analysis detected ‘Ca. P. pyri’ in a high proportion of insects, with maximum infection rates in March and April. These findings advance our understanding of ‘Ca. P. pyri’ spatial and temporal dynamics and their potential role in ‘Ca. P. pyri’ spreading under Chilean field conditions.

Abstract Delivery of therapeutic biomolecules into plant vascular tissues remains a challenge in management of vector-borne plant pathogens. The symbiont concept uses reprogrammed Agrobacterium tumefaciens galls (called symbionts) to produce biomolecules while remaining connected to host vasculature. We evaluated whether symbionts expressing antimicrobial peptides (AMPs) suppress ‘ Candidatus Liberibacter solanacearum’ (CLso), the causal agent of potato zebra chip disease. Symbionts were engineered to express a Streptococcus mutans bacteriocin associated with bacterial membrane disruption (Blp-Sm), or an AMP isolated from finger lime and associated with resistance to citrus greening disease (MaSAMP). Effects of AMP-producing symbionts on CLso titers, infection incidence, pathogen movement, and disease symptoms were evaluated in tomato and potato. In tomato, neither AMP significantly reduced CLso titers or infection incidence. However, in potato, AMP-producing symbionts reduced CLso accumulation and movement from CLso-inoculated source shoots into non-inoculated sink shoots connected through underground tubers. Blp-Sm produced the strongest reduction in CLso accumulation and infection incidence in sink tissues. In separate assays where symbionts were established directly on potato seed tubers, MaSAMP significantly reduced CLso titers in stems and tubers and reduced zebra chip symptoms in tubers, despite no reduction of CLso titers in terminal leaves. These findings demonstrate that AMP-producing symbionts suppress vascular pathogen accumulation and movement within plants and highlight the symbiont concept as a potential platform for managing diseases caused by vascular-restricted pathogens. Further, they show the potato-CLso system is a promising infection model to both refine and improve symbiont technology, and to test additional AMPs for potency against related pathogens.

Abstract Long-term agricultural conversion is known to shift soil microbial diversity and abundance in soils that formerly supported native grassland, but whether these shifts reflect uniform suppression across a bacterial genus or selective filtering of specific evolutionary lineages remains poorly understood. We addressed this question in Candidatus Udaeobacter, a globally abundant member of the phylum Verrucomicrobiota and a model oligotrophic soil bacterium. We collected 40 soil samples for RNA-Seq metatranscriptome analysis across three paired native prairie and long-term agricultural sites in Missouri and mapped transcriptional recruitment against a taxonomically curated consensus reference built from 36 concordant NCBI and GTDB Candidatus Udaeobacter genome assemblies. Total transcriptional recruitment to Candidatus Udaeobacter was nearly eleven-fold higher in prairie soils, and recruitment composition remained significantly distinct between land uses even after normalizing for this difference, indicating that land use reshapes which lineages remain active rather than uniformly reducing activity across the genus. This land use-associated recruitment showed strong phylogenetic signal, with closely related genomes exhibiting similar responses to land use. Genome architecture tracked this pattern and prairie-enriched lineages carried consistently smaller genomes and expressed a larger share of their coding capacity than agriculture-enriched lineages. These results show that environmental selection in Candidatus Udaeobacter operates below the genus level. Combining curated reference genomes with metatranscriptomic recruitment offers a scalable framework for resolving lineage-level ecological responses in other abundant, poorly characterized microbial taxa.

Scurfy pea (Cullen corylifolium (L.) Medik.) plants exhibiting symptoms of phyllody and witches’ broom indicative of phytoplasma infection were observed in the research field of ICAR – Indian Agricultural Research Institute, Regional Station, Pune (Maharashtra, India). Phytoplasma presence in all symptomatic samples was confirmed through PCR and nested PCR assay using phytoplasma-specific primer pairs P1/P7 and R16F2n/R2. BLASTn analysis revealed that the amplicon sequence shares more than 99% identity with known strains of ‘Candidatus Phytoplasma trifolii’ sequences. Phylogenetic analysis further clustered the sequence of the phytoplasma strain within the ‘Ca. Phytoplasma’ species while virtual RFLP analysis assigned it to the 16SrVI-D subgroup. To the best of our knowledge, this is the first worldwide report of 16SrVI-D phytoplasma associated with phyllody and witches’ broom in C. corylifolium.