Detection Of Five Citrus Diseases By RT-PCR And Multiplex RT-PCR

Citrus is one of the world's most important fruit crops in the world. China is one of the origin centres of citrus. Chinese people started to cultivate citrus fruits for commercial purposes long time ago. Now citrus is the second largest fruit crop in China. Citri-culture suffer in constant from increasing disease pressure, and great attention has been paid to control citrus disease. At Fruit Research Institute of Guangdong Academy of Agricultural Sciences from July 2004 to April 2006, studies were conducted for molecular indentification of Citrus tristeza virus (CTV) , Citrus Tatter Leaf Virus (CTLV), Citrus exocortis viroid (CEVd) , Huanglongbing (HLB) and Citrus Bacterial Canker Disease(CBCD). Reverse transcription polymerase chain reaction( RT-PCR ) was used to detect these diseases, a novel multiplex RT-PCR for simultaneous detection of multiple citrus diseases was established. The RT-PCR identification was reliable, rapid and sensitive for the detection of diseases.1. RT-PCR was optimized to detect CTV and CTLV. The special Taq and PCR enhancer were used to improve PCR of CEVd, which is of circular and single-stranded RNA with 59.5% of GC . A more sensitive RT-PCR for the detection of CEVd was established.2. RT-PCR for detection of HLB and CBCD was also reported. PCR for detection those five citrus diseases could apply at the same annealing temperature.3. A multiplex RT-PCR was established for simultaneously detection of two viruses of HLB and CTV. After optimizing the TaqDNA Polymerase , dNTPs, Mg²⁺, Primer and annealing. The best result was obtained with the PCR reaction containing Taq was 0.05U/μl, dNTPs was 0.25mmol/L, Mg²⁺ was 1.5mmol/L, Primers were HLB 0.30μmol/L and CTV 0.15 μmol/L and 63℃ for annealing.4. By use of an oligo(dT) 18 as a reverse transcription primer, ployadenylated RNAs viruses CTV and CTLV were detected by RT-PCR. We also found CEV could be detected in this way. CEV is a non-polyadenylated RNA viroid, but there is a fragment which is rich in A in the sequence of CEV. In the bases of uniplex RT-PCR, we developed a multiplex RT-PCR which could simultaneous detect CTV, CEV and CTLV. The results of the multiplex RT-PCR were consistent with uniplex RT-PCR, and permitted to detect each of the pathogens. The multiplex RT-PCR provides a simple and rapid method for detecting CTV, CEV and CTLV in citrus plants.