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Occurrence and molecular detection of huanglongbing in Bangladesh by monirul_hasan

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Occurrence and molecular detection of huanglongbing in Bangladesh

Background

Huanglongbing; HLB (citrus greening) is one of the emerging diseases of in citrus plants in Bangladesh and has been known in East Asia for over a century. The gram negative fastidious unculturable bacteria Candidatus Liberibacter asiaticus (Las) is the causal agent of huanglongbing. It is a destructive pathogen and widespread in Asia as well as in the Western hemisphere (Bové, 2006). It is spread by grafting and sap-sucking insect vector Asian citrus psyllid (Diaphorina citri Kuwayama) (Lin, 1963; Martinez & Wallace, 1967). Detection of Las is not easy because of its low concentration and irregular distribution in its citrus hosts (McClean, 1970). This disease is also difficult to be diagnosed by conventional procedures, whereas polymerase chain reaction (PCR) is the most convenient method for detection (Jagoueix et al., 1996).

 

Objectives

The study was undertaken to get an overview of the disease scenario; and to detect and identify the citrus greening causal organism, Candidatus Liberibacter asiaticus by PCR and sequencing.

 

Materials and Methods

Suspected leaves with characteristic irregular blotchy chlorosis symptoms were collected from major citrus growing areas of Bangladesh. 

The Las genomic DNA was extracted from midribs and petioles of symptomatic leaf using Wizard® Genomic DNA Purification Kit (Promega, Madison, USA) following manufacturer's instructions. 

The samples were then examined for the detection of Candidatus Liberibacter asiaticus by DNA-based polymerase chain reaction (PCR) using the ‘Las’ specific primers “A2” and “J5” to amplify a partial sequence of β-operon (rplKAJL-rpoBC operon) of ribosomal protein genes (Hocquellet et al., 1999). 

PCR products were sequenced directly in both orientations from primers A2/J5 according to the standard protocols for the ABI PRISM 3500 (version 3.7).

 

Results

A band of about 703 bp specific to Las strains was obtained after PCR amplification.

Partial nucleotide sequences of ribosomal protein genes of rplKAJL-rpoBC operon showed 99% sequence identity with Las strains found in India, China, Indonesia, and USA (GenBank accession # JQ964017, DQ157278, LC090233, and KR919751, respectively).

In the case of amino acid levels, it showed 97-98% protein sequence identity  identified with different isolated s deposits ed in GenBank (Accession # ACY26056, ABI35994, etc.).

Phylogenetic trees showed close relationships with Indian and Thai isolates.

 

Summary and Conclusion

The origin of Candidatus Liberibacter asiaticus in Bangladesh is uncertain.

We believe that it probably was introduced by infected material from the South Asian region. 

Results from PCR amplification and aligning ribosomal gene sequences gave strong evidence that the Las is present in Bangladesh.

It revealed the mystery of the citrus declining in Bangladesh which was associated with bacterial pathogen and not due to micronutrient deficiencies.