Polymerase chain reaction detection of Candidatus liberibacter asiaticus associated with citrus huanglonbing

Present studies in citrus orchards confirmed that Citrus Greening or Citrus huanglongbing (HLB), a heat sensitive citrus disease, is associated with the presence of “Candidatus liberibacter asiatic” in Maharashtra. It is associated with nonculturable phloem-restricted and gram-negative bacteria which is the members of the alpha subdivision of the proteobacteria. It is responsible for the decline of citrus orchards in Maharashtra and other citrus growing areas in the country. Molecular diagnosis of Candidatus liberibacter asiatic by using polymerase chain reaction which is used for detection of the disease when pathogen present is very low concentration in disease sample. Results revealed that among the three DNA isolation method viz., commercial kit method, sodium sulphite method and membrane bard nucleic acid technique, sodium sulphite method is cost effective for commercial use. The comparative cost of detection by various combination of reagent and sampling time was determined and cost effective technology was standardized and validated. The DNA extraction method by sodium sulphite, Klen Tag enzyme and 450 bp set of primers were identified to give best amplification in PCR system of diagnosis. The comparative cost of detection by various combination of reagent and sampling time was determined and cost effective technology was standardized and validated.