Molecular diagnosis of citrus greening bacterium

Abstract

The investigations on Citrus greening disease indicated that survey based on symptoms indicated 38.5 to 63.7% disease incidence in Andhra Pradesh and 20.0 to 86.7% incidence in Karnataka. The graft transmission experiments using greening affected scion material from Sweet Orange, Coorg Mandarin and Lime indicated that grafting successfully transmitted greening disease. In the case of Rangapur Lime rootstock 100% transmission was observed, with different scion materials, whereas 50 to 100% transmission was observed when Acid Lime, Troyer Citrange, Cleopatra Mandarin and Rough Lemon were used as rootstocks. In the grafted plants, symptoms appeared 7 to 9 weeks after grafting. Collecting psyllids, feeding on infected plants and transferring them onto susceptible plants successfully transmitted greening bacterium. Hundred percent transmission was achieved when more than 5 psyllids per plant were released and symptoms appeared after 2 to 4 months. Positive results were obtained from the DNA isolated from grafted plant samples and psyllids when subjected to PCR amplification. Among the bark, midrib and leaf lamina tested for the quality DNA for PCR analysis for CGB, bark tissue was found to be the best source for quality DNA followed by midrib. Even though DNA was successfully isolated from leaf lamina, it was not amplified when subjected to PCR analysis for CGB. This may be due to lack of phloem tissue in lamina as the CGB resides in phloem tissue. Among the fresh tissue and shade-dried tissues tested for quality DNA, the fresh tissue samples are found to be the best source. For PCR amplification, rDNA primers specific- to rDNA region successfully amplified 1.2 kb DNA fragment from CGB infected plant material, but not from healthy citrus plants or water as template indicating the specificity of the primers. Positive amplification of PCR confirming the presence of CGB was obtained in Coorg Mandarin, Rangapur Lime, Sweet Orange. Kinnow Mandarin, Acid Lime, Seedless Lime and Rough Lemon.