Polymerase Chain Reaction-Based Detection of Spiroplasma citri Associated with Citrus Stubborn Disease

Area of Science:

• Plant Pathology
• Molecular Biology
• Agricultural Science

Background:

• Citrus stubborn disease, caused by Spiroplasma citri, poses a significant threat to citrus production worldwide.
• Traditional diagnostic methods like culturing can be time-consuming and may lack sensitivity for field detection.
• Development of rapid and accurate molecular diagnostic tools is crucial for effective disease management.

Purpose of the Study:

• To improve polymerase chain reaction (PCR)-based detection of Spiroplasma citri using novel primers.
• To develop and evaluate a real-time PCR assay for enhanced sensitivity and quantification.
• To validate the performance of PCR methods against traditional culturing in field-collected citrus samples.

Main Methods:

• Designed and utilized new primers targeting the P89 and P58 genes of Spiroplasma citri for PCR assays.
• Developed a real-time PCR assay and determined its detection limits using serial dilutions of a recombinant plasmid.
• Compared PCR results with traditional culturing methods using DNA extracted from fruit columella of citrus trees in two orchards.

Main Results:

• PCR assays demonstrated high concordance (85-100%) with culturing results, with PCR detecting Spiroplasma citri in culture-negative samples (5-15%).
• Real-time PCR exhibited superior performance as the most effective detection method.
• Analysis of P58 primer pair reactions suggested the presence of two distinct Spiroplasma citri populations in field isolates.

Conclusions:

• The developed PCR and real-time PCR methods provide reliable and sensitive detection of Spiroplasma citri in citrus trees.
• PCR offers advantages over culturing, particularly in identifying infected trees that may be missed by traditional methods.
• The findings support the widespread adoption of PCR for accurate diagnosis and management of citrus stubborn disease.