Publications

4719

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.

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.

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 Ca. 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.

ABSTRACT Ponkan mandarin ( Citrus reticulata cv. Ponkan) is an important fruit crop planted across different countries. One of its most devastating diseases is citrus huanglongbing (HLB), which is associated with infection by the bacterium Candidatus Liberibacter asiaticus ( C Las). Although studies on HLB in Ponkan mandarin have been conducted, symptom heterogeneity and its association with C Las titer under field conditions remain underexplored. We examined the association between symptom variation and C Las infection status in a Ponkan mandarin grove by categorizing symptoms at both the leaf and canopy (tree) level. Chlorotic leaves were sampled and classified into different categories, including leaves exhibiting interveinal chlorosis (Cat. A), those showing vein yellowing (Cat. B), and blotchy mottled leaves (Cat. C). Polymerase chain reaction (PCR) and quantitative PCR (qPCR) assays targeting C Las revealed that Cat. A leaves, which resembled nutrient-deficient leaves, had higher C Las detection rates and a larger proportion of samples with high C Las titers. Canopy-level assessment of symptom diversity within each tree also revealed that plants bearing Cat. A leaves were more likely to carry C Las and have high pathogen loads. The likelihood was also greater in trees with higher leaf symptom diversity. Additional prophage typing of the detected C Las strains revealed that leaf symptom variations were independent of prophage type difference. Nutrient analyses revealed reduced magnesium, manganese, and zinc contents in Cat. A leaves, indicating that leaves prone to C Las infection also exhibited nutrient deficiencies. These findings highlight the complexity of potential cultivar-specific associations between C Las and citrus under field conditions. IMPORTANCE Although it is well-established that HLB in Ponkan mandarin is associated with C Las infection, the relationship between leaf symptom variation in this cultivar and the presence and titer of C Las under complex field conditions remains unclear. By examining symptom heterogeneity in natural field conditions at both the leaf and canopy level, this study demonstrated that HLB leaf symptom variation in naturally infected Ponkan mandarin corresponds to differences in C Las presence and titer at both levels. Notably, although blotchy mottling is considered the typical leaf symptom of HLB, we found that C Las detection rates and titers tended to be greater in leaves showing nutrient deficiency-like symptoms, which contrasts with patterns in other citrus cultivars. Recognizing inconsistencies among cultivar-specific patterns is important for interpreting HLB symptoms, devising sampling strategies, and exploring uncharacterized factors affecting symptom expression under field conditions.

Abstract A new distribution map is provided for Candidatus Phytoplasma rubi Malembic-Maher et al. Mollicutes: Acholeplasmatales: Acholeplasmataceae. Hosts: Rubus spp.