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

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.