Publications

4862

Huanglongbing (aka: citrus greening) associated with ‘Candidatus Liberibacter asiaticus’ (CLas) is one of the most impactful citrus diseases throughout the world. The aim of this project was to support the sensitive and rapid detection of CLas to prevent the continued spread and destruction of US citrus crops by this psyllid vector-borne disease. Two multicopy targets specific to CLas were selected for assay development: the five-copy nrdB (ribonucleotide reductase-RNR) gene and a four-copy locus 4cp (4CP). Published RNR RPA primers were used to develop a one-pot RPA-CRISPR assay, and RPA primers for 4CP were designed. crRNAs for both RNR- and 4CP-RPA-CRISPR/Cas assays were designed, evaluated through preliminary testing, and the best candidate primers and crRNA combinations were selected. The 4CP assay was selected and subjected to a battery of validation tests and determined fit-for-purpose. A field extraction protocol was also developed using commercial reagents, syringes and filters to bring kit sensitivity to the field. When combined with the assay, the complete workflow was able to detect CLas in infected citrus, down to a single infected leaf per subsample; the field workflow showed similar sensitivity to the laboratory-based workflow. While the extraction and assay proved useful, particularly for symptomatic leaves, its performance with asymptomatic leaves for early detection was not evaluated. The complexity of the assay in its current form hinders widespread practical applicability. In the future, revalidation of lyophilized reagents in a convenient kit can be performed for practical deployment; preliminary testing has shown success.

The phloem-colonizing bacterial pathogen Candidatus Liberibacter asiaticus (CLas) causes the devastating citrus Huanglongbing (HLB). Despite the important roles of roots, how CLas affects roots remains largely unknown. Here we dissected host responses in leaf and root at different time points after CLas inoculation. Our data showed that CLas triggers less reactive oxygen species (ROS) accumulation and phloem callose deposition, which are known to contribute to HLB symptom development in leaves, in roots than leaves. Increased chloroplastic ROS accumulation was detected in CLas-infected leaf compared to uninfected controls, which may explain the increased ROS levels in leaves than roots infected by CLas. qRT-PCR and RNA-seq analyses showed global induction of immune related pathways in both roots and leaves, whereas photosynthesis related pathways were only downregulated in leaves, but not in roots, by CLas infection. The differential expression pattern related to immunity and photosynthesis in the leaf and root tissues suggests that chloroplasts might be involved in the differences in ROS accumulation and callose deposition. Root cell death was observed in HLB symptomatic trees regardless of CLas infection status of roots. Significantly more starch accumulation was first observed in CLas positive leaves than healthy controls followed by starch depletion in CLas positive roots later, suggesting a causative relationship between them. Taken together, root cell death caused by CLas infection maybe caused by carbon starvation rather than via a similar mechanism as in leaves. Our data suggests chloroplasts play a critical role in the different pathogenicity mechanisms of CLas in leaf and root.

Huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (CLas), poses a major threat to citrus production worldwide and has recently emerged as a serious constraint to sweet orange cultivation in Bangladesh. Despite widespread disease occurrence, information on the genetic diversity of CLas populations in the country is lacking. This study investigated the genetic structure of CLas across major sweet orange–growing regions of Bangladesh using tandem repeat number (TRN) variation at the CLIBASIA_01645 genomic locus. Field surveys were conducted in 17 districts between 2022 and 2023, and 89 HLB-symptomatic leaf samples were collected from commercial orchards. PCR and qPCR detection confirmed CLas infection in 67 (75%) and 74 (83%) samples, respectively. Thirty-seven representative isolates were sequenced and analyzed for TRN polymorphism. Ten TRN genotypes (TRN2–TRN12, excluding TRN3) were identified, with TRN6 being the most prevalent. Bangladeshi CLas isolates were further grouped into three classes based on TRN: Class I (TRN ≤ 5), Class II (5 < TRN ≤ 10), and Class III (10 < TRN ≤ 15), of which Class II was the predominant. All Bangladeshi isolates harbored the CTTGTGT repeat motif and clustered closely with Indian isolates in haplotype network and phylogenetic analyses, while isolates carrying the AGACACA motif from other countries formed a distinct clade. These results reveal intraspecific diversity of CLas in Bangladesh and suggest regional structuring and shared ancestry with neighboring Indian populations. This study provides the first population-level genetic insight into CLas in Bangladesh and establishes a foundation for improved epidemiological understanding and disease management.

Abstract Synthetic endosymbiosis, the deliberate introduction of bacteria into eukaryotic host cells, provides a tractable model for how endosymbiotic relationships become established, and a route to cells with new metabolic capabilities that never evolved naturally in that lineage. Current methods for introducing bacteria into mammalian cells rely on microinjection, engineered invasion, listeriolysin-mediated phagosomal escape, or naturally permissive hosts. These methods are invasive, low-throughput, or costly, and most require the prospective endosymbiont to be genetically tractable, which excludes endosymbiotic bacteria which are of great interest as potential organelle precursors. Here we describe a protocol based on polyethylene glycol (PEG), long used to fuse mammalian cells, that delivers Escherichia coli into HeLa cells without genetic modification of either partner or the use of specialised equipment. We optimised the governing parameters in two stages: first, bacterial density, gentamicin concentration, and PEG molecular weight and concentration were optimised; then, medium composition and the timing of fusion and recovery. Across four parameters screened, 10% PEG-3350 applied for 4 min to HeLa cells 12 h after seeding, followed by 4 h in gentamicin-containing medium, gave the most consistent delivery. Under these conditions, confocal microscopy and z-stack reconstruction detected mCherry-tagged E. coli inside HeLa cells. Applying the same protocol to Candidatus Kinetoplastibacterium crithidii, a naturally occurring β-proteobacterial endosymbiont of the trypanosomatid Angomonas deanei , we observed the endosymbiont inside the HeLa cells. This protocol offers an accessible entry point for constructing artificial endosymbioses.

Abstract Bacterial symbionts were shown to have diverse but significant impacts on the fitness of their host in insects, but little is known on the symbionts of spiders and their interactions with their hosts. Here, we assembled and investigated the circular 575 kb genome of Candidatus Argiopiplasma dusa, a novel bacterial symbiont of the wasp spider Argiope bruennichi. Phylogenomic analysis placed this species within the phylum Mycoplasmatota, in a poorly characterized clade that may represent a new order-level lineage or affiliate with the Mycoplasmatales order. With 559 predicted genes, the genome is relatively small compared to other Mycoplasmatota genomes (on average 969 genes) and has a low GC content of ∼24%. While the genome encodes genes for proteins involved in glycolysis and fermentative acetate production, it revealed minimal biosynthetic capabilities with pathways for nucleotide, amino acid and vitamin biosynthesis being absent in Ca. Argiopiplasma dusa. This suggests a metabolic dependence and an endosymbiotic lifestyle within the spider host. The symbiont was detected in A. bruennichi populations across the distribution range of the spider but appears to be absent in certain populations.

Early and accurate detection of ‘Candidatus Phytoplasma pruni’ is essential for the control of X-disease in Prunus species, but this is made difficult by pathogen biology and the ability to consistently recover nucleic acids, both of which increase the likelihood of false negatives. Here we examined the latter, using high- and low-titer infected sweet cherry (P. avium) samples to compare three tissue disruption methods, four nucleic acid extraction chemistries, and four PCR chemistries. Results showed that method selection had minimal impact on high-titer samples but significantly affected low-titer detection. Bead-beating disruption consistently outperformed rotary and manual grinding, yielding lower Cq values and more reliable amplification. Among extraction methods, CTAB-based protocols provided the highest nucleic acid recovery and detection sensitivity, particularly when paired with bead beating. Column- and magnetic bead–based kits performed less consistently, especially with low-titer samples, where false negatives were frequent. PCR chemistry also influenced results, although RT-qPCR did not significantly improve sensitivity over qPCR. Importantly, commonly used host internal controls failed to reliably indicate pathogen detection success, as high-abundance targets masked poor extraction efficiency. Cumulatively these data suggest the need for optimizing diagnostic protocols to ensure capture of low-concentration pathogens to avoid false negatives.