Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.09.19.677271

PCR-Based Detection and Phylogenetic Analysis of Candidatus Liberibacter asiaticus in Citrus Orchards Across Nepal

Oliya, B. K.·Giri, R.·Gautam, S. G.·Manandhar, K. D.
View original source ↗Export metadata ↓

Abstract

Citrus greening disease, also known as huanglongbing (HLB), is caused by the gram-negative -proteobacteria Candidatus Liberibacter species. This disease poses a significant threat to citrus production worldwide, including in Nepal. This study aimed to conduct the diagnosis and phylogenetic analysis of the citrus greening pathogen in Nepal using both conventional PCR and computational methods. A total of 1,026 samples were collected from thirteen districts across six provinces in the country. PCR-based diagnosis was performed using the primer set Las606/LSS, which targets the 16S rRNA gene of Candidatus Liberibacter asiaticus. Additionally, 16S rRNA gene sequencing was performed using sanger sequencing for five samples collected from different geographical regions. The obtained sequences were deposited in GenBank, and a phylogenetic tree was constructed based on these sequences. Among the 1,026 samples tested, 255 were positive, indicating the widespread distribution of HLB across Nepal. All consensus sequences from Nepal showed strong evolutionary relatedness within the C. L. asiaticus cluster, displayed over 99% genetic similarity with reference sequences from various parts of the world. Phylogenetic analysis revealed that the Nepalese sequences were closely related to C. L. asiaticus sequences from India, such as OM522080.1 (Punjab) and MH473394.1 (Meerut). Additionally, sequences obtained from different regions of Nepal clustered closely together. The findings of this research provide valuable insights into the genetic diversity and prevalence of citrus greening disease, which will aid the development of better management strategies to combat this devastating disease. Furthermore, the sequence data deposited in GenBank will serve as a key reference for future molecular studies on citrus greening disease.

Source connections

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Oliya, B. K., Giri, R., Gautam, S. G., Manandhar, K. D.. 2025-09-19. PCR-Based Detection and Phylogenetic Analysis of Candidatus Liberibacter asiaticus in Citrus Orchards Across Nepal. https://doi.org/10.1101/2025.09.19.677271

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Hidden Biodiversity in Wildlife Trade Networks: DNA Barcoding Reveals Fish and Crocodilian Species in Commercialized Swim Bladders

International wildlife trade represents one of the major drivers of biodiversity exploitation worldwide. However, the true taxonomic diversity embedded within commercial wildlife products often remains unknown because processing removes diagnostic morphological characteristics, preventing reliable species identification. Consequently, biodiversity assessments based solely on product labels may substantially underestimate the diversity of species involved in trade networks. To investigate hidden biodiversity within wildlife trade products, we applied DNA barcoding based on the mitochondrial cytochrome c oxidase subunit I (COI) gene to 77 products commercialized as fish swim bladders and seized at Guarulhos International Airport, Brazil. Molecular analyses successfully identified all samples and revealed the presence of four species: Plagioscion auratus (n = 38), Cynoscion acoupa (n = 7), Melanosuchus niger (n = 17), and Caiman crocodilus (n = 15). Fish species accounted for 71.4% of all samples, whereas crocodilians represented 28.6%, demonstrating that products marketed under a single commercial category may conceal substantial taxonomic diversity. Notably, the occurrence of two Amazonian crocodilian species within a trade chain traditionally associated with fish products reveals a previously undocumented component of the international wildlife trade. Our findings demonstrate that DNA barcoding is an effective tool for uncovering hidden biodiversity within processed wildlife products and provide evidence that wildlife trade networks may involve a broader spectrum of species than suggested by commercial labels. These results highlight the importance of molecular surveillance for biodiversity monitoring, wildlife trade regulation, and conservation planning.

molecular biology↗
bioRxiv2026

Plasmid architecture determines the stability of inverted terminal repeats in adeno-associated virus vectors

Recombinant vectors derived from adeno-associated viruses (rAAVs) are a mainstay of human gene therapy. rAAVs are produced from plasmids containing transgene cassettes flanked by inverted terminal repeats (ITRs), which form structured DNA elements that stabilize the ends of the single-stranded viral genome and are the only viral sequences required in cis for genome packaging. For decades, it has been recognized that propagation of ITR-containing plasmids can result in deletions and other mutations, prompting the use of specialized bacterial strains, modified growth conditions, and truncated or altered ITRs. Despite these practices, ITR instability remains a persistent source of plasmid heterogeneity. To identify determinants of ITR stability, we evaluated ITR integrity in one of the original cloned AAV2 genome isolates, a reconstructed AAV2 plasmid, and a synthetic rAAV vector containing full-length native AAV2 ITRs. We established a quantitative bioinformatic workflow for analyzing ITR-containing plasmids and virus preparations from raw Oxford Nanopore sequencing data. These experiments showed that ITRs were highly stable during short-term culture, whereas prolonged culture revealed strong positional effects, with preferential loss or mutation of the ITR nearest the plasmid origin of replication. Consistent with this model, a survey of 7,041 sequence-verifiable AAV plasmids from the Addgene repository identified a widely disseminated 11-bp ITR deletion in 4,773 plasmids; among analyzable two-ITR plasmids, this deletion was located in the origin-proximal ITR in 95.3% of cases. Guided by these findings, we constructed a novel rAAV entry vector with stable full-length native AAV2 ITRs that enabled efficient packaging of a 4,750-bp all-in-one CRISPR-Cas9 cassette. Finally, we developed a cell-based strategy to compare the effects of ITR mutations on rAAV genome integration, providing preliminary evidence that ITR sequence variation can influence integration outcomes. Together, these findings show that ITR instability is a preventable, position-dependent property of plasmid architecture and identify ITR integrity as an important variable in rAAV vector design and quality control.

molecular biology↗
bioRxiv2026

Single-point mutation alters odorant receptor sensitivity associated with host plant specialization in Spodoptera moths

Host specialization in herbivorous insects is often associated with divergence in chemosensory abilities. Here, we investigated the possible contribution of odorant receptors (ORs) in host plant restriction in the lily moth Spodoptera picta, a species specialized on Amaryllidaceae. Manual annotation of S. picta ORs in its genome revealed a repertoire similar in size and composition to those of its polyphagous sister species, S. littoralis and S. litura, suggesting that specialization did not involve major gene loss or expansion in the lily moth. To assess functional divergence beyond gene number, we applied a large scaled structure-based virtual screening approach to the entire OR repertoires of these three Spodoptera species, generating ligand-binding profiles for 120,591 volatile compounds. Among 69 1:1:1 OR orthologs, 24 exhibited divergent predicted binding spectra. We pinpointed OR29 that we also found to be highly expressed in both male and female antennae of S. picta through a RNAseq approach. Functional assays demonstrated that S. picta OR29 acquired heightened sensitivity to limonene enantiomers, volatiles emitted by host Amaryllidaceae inflorescences. Site-directed mutagenesis revealed that a single amino acid substitution within the predicted binding region underlies this shift in sensitivity. These results show that host specialization in S. picta has not been accompanied by significant OR repertoire remodeling, but rather by subtle molecular changes that fine-tune receptor sensitivity to host-derived volatiles.

molecular biology↗