Revisiting Citrus Greening Surveillance in Nepal: Molecular Evidence for Diagnostic Reform

ABSTRACT

Huanglongbing (HLB), associated with ‘ Candidatus Liberibacter asiaticus’ ( C Las), poses an escalating threat to citrus production in Nepal, where yields declined by 5.6% in 2021/22 compared to 2019/20 and old orchards have decreased by ~11% over the past decades. Despite these losses, national surveillance depends largely on conventional PCR without local validation. We conducted the first nationwide molecular evaluation of C Las, analysing 97 samples (1552 individual leaves) from 11 major citrus‐producing districts using conventional PCR, TaqMan‐based quantitative PCR (qPCR) and 16S rRNA gene sequencing. The commonly used OI1/OI2c primer detected only 26.8% of infections, whereas Las606/LSSR and qPCR increased the detection rate to 69.1% and 85.6%, respectively. Detection efficiency was strongly associated with symptom severity ( p  < 0.0001 for all assays). Notably, qPCR significantly ( p  = 0.002) outperformed Las606/LSSR in leaf samples with no typical visual symptoms of infection, highlighting substantial early stage infections overlooked by conventional methods. Bacterial loads ranged from 1.1 × 10 ¹ to 6.6 × 10 ⁶ copies/g. Sequence analysis revealed a single, genetically homogeneous C Las lineage across the country, closely related to Indian and Chinese strains, implying one historical introduction followed by domestic spread. These findings demonstrate that Nepal's current surveillance underestimates infection prevalence, particularly at early stages. While conventional PCR suits basic surveillance and quarantine, we recommend incorporating routine qPCR, at least biannually, for nursery certification and mother‐stock screening. This study establishes the first molecular baseline for C Las diversity and pathogen load in Nepal, providing critical evidence to guide national HLB management and policy reform.