Identification and Molecular Characterization of Candidatus Liberibacter asiaticus (Citrus Huanglongbing Disease Pathogenic Agent) and Its Control by Plant Extracts in Bangladesh

Aims: To identify and characterize Citrus Huanglongbing disease causing pathogen Candidatus Liberibacter asiaticus and to evaluate its biological control using medicinal plant extracts.

Study Design: The study was designed based on standard laboratory protocol.

Place and Duration of Study: Professor Joarder DNA and Chromosome Research Lab, Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh between June 2015 and September 2016.

Methodology: Causal pathogen of Citrus Huanglongbing disease was isolated from infected leaves. Different types of biochemical and morphological characterizations of Candidatus Liberibacter asiaticus were done. 16S rDNA primers (27F and 1391R) were used to amplify genomic DNA of Candidatus Liberibacter asiaticus. Sequencing of 16S rDNA sequence of Candidatus Liberibacter asiaticus were performed. Sensitivity pattern of Candidatus Liberibacter asiaticus against several standard antibiotics were done. Antimicrobial activity test was observed using two solvents extracts of four medicinal plants by disc diffusion method in vitro condition.

Results: Candidatus Liberibacter asiaticus showed positive and negative response to different biochemical test. Candidatus Liberibacter asiaticus showed gram negative in gram staining test. Candidatus Liberibacter asiaticus showed highest 35±0.5 mm and lowest 8±0.2 mm zone of inhibition against amoxycillin and kanamycin respectively. Approximately 1300bp band was found in PCR amplification and phylogenetic tree analysis of 16S rRNA gene sequence of Candidatus Liberibacter asiaticus showed 75% similarity with Candidatus Liberibacter asiaticus strain 374.15. Candidatus Liberibacter asiaticus showed highest 16±0.5 mm diameter of zone of inhibition at 60 µg/ml concentration for ethanol extract of Cuscuta reflexa.

Conclusion: This study will be helpful for proper identification, characterization and control of Citrus Huanglongbing disease causing pathogen Candidatus Liberibacter asiaticus in an eco-friendly way.