Publications

4954

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 Background The Toll signaling pathway is a key component of insect innate immunity, yet its role in hemipteran vectors and their interactions with plant pathogens remains poorly understood. The potato psyllid ( Bactericera cockerelli ), an important agricultural pest in the USA, transmits two haplotypes of ‘Candidatus Liberibacter solanacearum ’ (LsoA and LsoB) that cause severe diseases in solanaceous crops. Methods and Results We characterized Toll pathway components in the B. cockerelli genome and examined their transcriptional responses during pathogen acquisition. Six Toll receptors and ten pathway-associated genes were identified in the psyllid genome. Gene expression was evaluated using RT-qPCR following exposure to the entomopathogenic fungus Beauveria bassiana (Bb-490) and infection with Lso haplotypes. The transcription of five Toll receptors was significantly induced in response to B. bassiana , indicating their potential role in antifungal defense. In contrast, Lso infection elicited haplotype-specific transcriptional responses: two Toll receptors (Toll like-1 and Toll-7) were up-regulated during LsoB acquisition, whereas only Toll-7 responded to LsoA. In addition, Spätzle-3 was induced by LsoB, while Spätzle was induced by LsoA. Persistent Lso infection did not significantly alter the transcriptional expression of Toll receptors. Conclusions These findings provide new insights into immune regulation in B. cockerelli and highlight the potential of targeting Toll-mediated responses and fungal biocontrol strategies for sustainable disease management.

Veracruz, one of Mexico’s main citrus-producing regions, faces a major phytosanitary threat from huanglongbing (HLB) because of its rapid spread and atypical symptom expression. Symptom variability limits the reliability of field diagnosis and highlights the need to correlate visible symptoms with molecular detection of Candidatus Liberibacter asiaticus (CLas). This study aimed to evaluate the qPCR positivity rate for CLas in symptomatic citrus samples collected from seven citrus-producing municipalities in Veracruz. Branch dieback was the most frequent symptom (67.5 %), followed by generalized yellowing (20.4 %). The qPCR positivity rate in symptomatic CLas samples ranged from 94 % in Chicontepec to 98.7 % in Ixhuatlán de Madero and Castillo de Teayo. Fruit ripening, asymmetric fruit development, and irregular spots on orange leaves cannot be considered classic symptoms of HLB in northern Veracruz because they do not occur recurrently. Logistic regression analysis (odds ratios [OR]) showed no significant association between qPCR positivity and municipality, cultivar, or rootstock. In contrast, generalized yellowing was significantly associated with higher odds of CLas qPCR positivity (OR = 2.29, p = 0.001) (an OR > 1 indicates a higher risk). These results indicate that generalized yellowing is the most strongly associated symptom with CLas infection in citrus trees in northern Veracruz.

ABSTRACT This study investigated the acclimatization of Candidatus Brocadia, a freshwater anaerobic ammonium oxidation (Anammox) species, to high nitrogen (up to 1.39 gN/L) and salinity (up to 14.3 gNaCl/L) loads in a semicontinuous sequencing batch reactor. Over one year of operation, the reactor achieved stable nitrogen removal performance, with nitrite removal efficiency exceeding 99% at nitrogen loading rates and salinity concentrations up to 0.72 gN/L/d and 14.29 gNaCl/L, respectively. These results rank among the highest reported for Candidatus Brocadia under similar conditions, demonstrating the feasibility of applying this freshwater species to high-strength, saline effluents such as those from agricultural systems. Batch experiments were conducted to further explore the impact of salinity and the monovalent-to-divalent cation ratio on Anammox activity. The results suggest that while salinity is the primary inhibitory factor, the presence of divalent cations such as Ca2+ and Mg2+, which likely mitigate salinity, may explain the high performance achieved in this study. Additionally, a novel method for real-time determination of nitrite consumption kinetics via pH monitoring was validated, offering a reliable tool for Anammox activity assessment.

The pathogen Candidatus Liberibacter asiaticus (CLas) is the cause of Huanglongbing (HLB) or citrus greening (CG) disease, which is one of the devastating diseases causing a significant crop reduction in citrus crops (Family: Rutaceae). It is a systemic pathogen which lives in citrus crop phloem tissues. Detection of Ca. L. asiaticus (CLas) casuing citrus Huanglongbing (HLB) disease was facilitated by taking samples from three different citrus orchards located in Tirupattur, Dharmapuri, Krishnagiri district. Midrib injection method by using syringe needle or microneedles used has early transmission of Ca. L. asiaticus (CLas) in phloem tissue of acid lime leaves. Total genomic DNA is extracted from inoculation of citrus leaves using a modified CTAB method to identify DNA from both healthy and infected samples. A 100 mg leaf midrib sample was crushed in liquid nitrogen, homogenized in 500 µl CTAB, incubated for 45 minutes at 60°C in a water bath, centrifuged for 5 minutes at 12000 rpm, and the supernatant was then transferred to a fresh tube, to which 5 µl of RNase-A was added, and incubated for 20 minutes at 37°C. For phase separation, an equal volume of chloroform/isoamyl alcohol (24:1) was added, vortexed for five minutes, and centrifuged for two minutes at 12000 rpm. The upper phase was then transferred to a new tube, in which 500µl of chilling isopropanol was added, kept at -20°C for 15 minutes, centrifuged at 12000 rpm, and the pallet was cleaned twice with 70% ethanol. The specific primers OI1/OI2c and A2/J5 had been used in a polymerase chain reaction (PCR) for detection, and they showed a specific band of 1160 bp and 703 bp, respectively. These were amplified from diseased leaves whereas no product from healthy citrus plant.

ABSTRACT The genus Rickettsia comprises obligate intracellular bacteria belonging to the class Alphaproteobacteria. Although Rickettsia species have traditionally been classified into several phylogenetic groups, recent genome-based studies have revealed greater diversity within the genus and related Rickettsiaceae lineages than previously recognized. Here, we isolated six rickettsial strains from Ixodes turdus ticks collected at various locations across Japan and performed whole-genome sequencing. A closed-genome sequence was obtained for one of the isolates. The six isolates shared highly similar genomic features and were identified as “ Candidatus Rickettsia mendelii” on the basis of conserved rickettsial marker gene sequences. The genomes of “ Ca . R. mendelii” were smaller (1.05–1.06 Mb) and had lower GC content (29.2–29.3%) than most analyzed rickettsial genomes and were comparable in these respects to those of Typhus Group rickettsiae. Family-wide phylogenomic analysis using a quality-filtered Rickettsiaceae genome data set placed “ Ca . R. mendelii” in a distinct early-diverging lineage within the genus Rickettsia . Chromosome-level comparisons with many other Rickettsia species revealed stretches of conserved synteny distributed across the chromosome of “ Ca . R. mendelii.” Genes encoding major secretion-related and host interaction-associated components were detected, whereas no hits meeting the detection criteria were obtained for the F-type IV secretion system (F-T4SS) proteins encoded by Rickettsiales-amplified genetic elements (RAGEs). These results provide the first genome sequences from cultured “ Ca . R. mendelii” isolates and expand our understanding of genome diversity and evolution within the genus Rickettsia . IMPORTANCE Genome-based studies have expanded the known diversity of the genus Rickettsia and related Rickettsiaceae lineages, but genome information from cultured isolates remains unavailable for some poorly characterized taxa. Here, we report the first whole-genome sequences from six cultured isolates of “ Ca . R. mendelii” and show that this taxon represents a distinct early-diverging lineage within the genus Rickettsia . The genomes of “ Ca . R. mendelii” were smaller and had lower GC content than most analyzed rickettsial genomes and were comparable in these respects to those of Typhus Group rickettsiae, despite the distinct phylogenetic placement. This indicates that similar genome characteristics can occur in phylogenetically distant rickettsial lineages. The low genomic diversity among geographically separated “ Ca . R. mendelii” isolates suggests broad dispersal of closely related strains, which may be facilitated by infected ticks carried by migratory birds. These findings improve our understanding of the genome characteristics and geographic distribution of “ Ca . R. mendelii.”

Anopheles plumbeus is a widespread forest mosquito species and an effective vector of human, animal, and avian malaria pathogens. In this study, we sequenced and assembled the genome of female An. plumbeus mosquitoes obtained from a laboratory culture. The mosquitoes were collected at the larval stage in the vicinity of Sochi (Black Sea coast of the Caucasus) and reared to adulthood on an artificial diet. The genome was generated using Oxford Nanopore Technologies long-read sequencing. The resulting hologenome comprises 1,449 contigs with a total length of 188,457,274 bp. It has been deposited in the European Nucleotide Archive (ENA) and is publicly available as a metagenome-assembled genome. The high quality of the eukaryotic metagenome-assembled genome (eMAG) assembly was confirmed by BUSCO analysis, demonstrating 97.5% complete BUSCOs. A single intracellular bacterial symbiont, Candidatus Tisiphia sp., was identified within the hologenome. The symbiont genome was assembled via a Whole-Genome Shotgun (WGS) approach using the Flye assembler (v. 2.9-b1774), yielding a size of 1.7 Mb. The registered assembly of Candidatus Tisiphia sp. was designated as a 'reference sequence'. Genome annotation revealed 1,761 genes, including 1,525 protein-coding genes, 4 rRNA genes, and 38 tRNA genes. A distinctive feature of this novel Candidatus Tisiphia genome is the high abundance of IS DNA transposons belonging to several families. In total, 361 IS elements were identified in the Candidatus Tisiphia sp. genome. Eight identified IS transposon families—IS6, IS701, IS4, IS630, ISNCY, IS110, IS5, and IS1634—are multicopy. Crucially, we identified for the first time in this bacterial species the biosynthetic genes for folic acid (vitamin B9), biotin (vitamin B7), and thiamine (vitamin B1), as well as a gene cluster for heme biosynthesis, which is essential for the functioning of mitochondrial cytochromes in the mosquito's respiratory chain. These data provide a foundational framework for developing biocontrol strategies against this potentially dangerous malaria vector.

ABSTRACT ‘ Candidatus Phytoplasma solani’ is a widespread and epidemiologically important phytoplasma in Europe with a broad host range, including sugar beet, potato and grapevine. In sugar beet, the recently emerging 16SrXII‐P subgroup of ‘ Ca . P. solani' frequently co‐occurs with the phloem‐associated bacterium ‘ Candidatus Arsenophonus phytopathogenicus’, which is considered the primary causal agent of the ‘syndrome des basses richesses’ (SBR), a disease that significantly affects yield and sugar content in central Europe. Currently, diagnosis and differentiation of ‘ Ca . P. solani' rely on real‐time quantitative PCR (qPCR) and nested PCR, which are labour‐intensive, costly and require complex instrumentation. Therefore, we developed a loop‐mediated isothermal amplification (LAMP) assay for specific detection of 16SrXII‐P phytoplasma that is suitable for rapid laboratory use and potential on‐site testing using crude plant and insect extracts. The new LAMP assay targets the stamp gene of 16SrXII‐P and was evaluated together with a published LAMP assay targeting the secA gene of ‘ Ca . P. solani' 16SrXII‐A. This combination enabled reliable detection and discrimination of 16SrXII‐P and 16SrXII‐A phytoplasma in sugar beet tissues and in the vector Pentastiridius leporinus . The 16SrXII‐P‐specific LAMP assay showed high analytical specificity and showed comparable performance to a TaqMan qPCR assay in the reliable detection of positive samples. In combination with simple NaOH‐Tris and phosphate‐buffered saline (PBS)‐based crude extraction protocols, the assay provided clear positive or negative results within 30 min, making it a practical tool for surveillance of 16SrXII‐P phytoplasma and rapid differentiation between the two ‘ Ca . P. solani' subgroups infecting sugar beet.