Title : Citrus greening disease and its molecular variavility
Citrus is one of the most economically important fruit crops in India. Commercially grown citrus includes sweet orange, acid lime and mandarin citrus greening bacterium (CGB), a bacterial disease are the two most important diseases that are impending fruit production through the world. Symptoms include yellow shoots, , smaller or lopsided fruits with aborted seeds, root degradation and tree decline HLB affects all commercial citrus varieties and a source of genetically resistant germplasm remains unknow. Despite these efforts, the most effective way to control HLB would be the replacement of susceptible citrus varieties by HLB-resistant plants. However, no resistant trees or scion-rootstock combinations have been identified so far. Citrus genetic transformation via the introduction of a single trait is an opportunity for improvement of citrus varieties, maintaining their genotypic and phenotypic characteristics. Different genetic strategies have been tested to obtain HLB-resistant citrus varieties.
The total genomic DNA was isolated from midrib of leaf of greening infected Kinnow mandarin and healthy leaf of kinnow mandarin plants by the method of DNeasy plant mini kit method (Qiagen Gmbh, Hilden, Germany) as described in earlier. primers on 16S, 23S and 16S/23S intergenic region of citrus greening bacterium were design.
Cloning of amplified products and transformation were done. Colony PCR was employed to confirm positive clones. Transferred small amount of bacterial cells from selected recombinant colonies (Two to ten) cells from individual grids were using sterile toothpicks to PCR tubes (200 µl) containing the PCR mixture bacterial cells served as template in PCR mix. Amplification conditions described earlier were followed and PCR products were electrophoresed in 1.0% agarose gel containing ethidium bromide. Sequences of 16S rRNA gene, 16S/23S Intergenic spacer region and 23S rRNA of greening bacterium associated with kinnow mandarin was determined for the first time.The intergenic spacer region sequence of different species of greening bacterium was highly variable compared to 16S rRNA and 23S rRNA sequences indicating that intergenic region can be used for taxonomic differentiation of greening bacterium