Development of Rapid, Sensitive and Non-Radioactive Tissue-Blot Diagnostic Method for the Detection of Citrus Greening

Citrus Huanglongbing (HLB) also known as citrus greening, is one of the most devastating disease of citrus worldwide. The disease is caused by Gram-negative, phloem limited α-proteobacterium, ‘Candidatus Liberibacter asiaticus’, vectored by the psyllid, Diaphorina citri Kuwayama. Citrus plants infected by the HLB bacterium may not show visible symptoms sometimes for years following infection and non-uniform distribution within the tree makes the detection of the pathogen very difficult. Efficient management of HLB disease requires rapid and sensitive detection early in the infection followed by eradication of the source of pathogen and the vector. The polymerase chain reaction (PCR) based method is most commonly employed for screening the infected/suspected HLB plants and psyllids. This is time consuming, cumbersome and not practical for screening large number of samples in the field. To overcome this, we have developed a simple, sensitive, non-radioactive, tissue-blot diagnostic method for early detection and screening of HLB disease. Digoxigenin labeled molecular probes specific to ‘Candidatus Liberibacter’ nucleotide sequences have been developed and used for the detection of the pathogen of the HLB disease. The copy number of the target genes were also assessed using real-time PCR experiments and the optimized real-time PCR protocol allowed positive HLB detection in citrus samples infected with HLB bacterium.