|MAHESHWARI, YOGITA - Foreign Agricultural Service (FAS, USDA)|
|SELVARAJ, VIJAY - Foreign Agricultural Service (FAS, USDA)|
|HAJERI, SUBHAS - Central California Tristeza Eradication Agency|
|Yokomi, Raymond - Ray|
Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 5/17/2017
Publication Date: 12/1/2017
Citation: Maheshwari, Y., Selvaraj, V., Hajeri, S., Yokomi, R.K. 2017. Detection of Spiroplasma citri by droplet digital PCR. Phytopathology. 107:S5:58.
Technical Abstract: Spiroplasmas are motile, helical bacteria belonging to the Class Mollicutes, a group of prokaryotics with no cell wall and phylogenetically related to Gram-positive bacteria. Spiroplasma citri is the first-cultured spiroplasma and causal agent of citrus stubborn disease (CSD). Detection of CSD is difficult due to low and erratic titer and distribution of the pathogen in infected tissues and isolation by culturing is technically demanding and time consuming. This study evaluated the applicability of droplet digital PCR (ddPCR) as an improved new tool with higher accuracy and precision for detection of S. citri at low titer and, presumably, at an early stage of infection using two sets of primers SP1 and ORF1 for spiralin (housekeeping) and prophage (multi-copy) genes, respectively. Standard curve analyses on tenfold dilution series of S. citri cells showed that ddPCR assay had good linearity and PCR efficiency. The sensitivity of ddPCR assay for SP1 and ORF1 plasmids were 1 copy/20µl reaction and 3.4 copies/20µl, respectively. The sensitivity of ddPCR assay with S. citri DNA was 0.00001 ng using SP1 and ORF1. Leaves and fruit samples were collected from different quadrant branches from fifty trees in a citrus orchard near Ducor, Tulare County, CA. The ddPCR assay showed that S. citri titer was higher in fruit columella compared to leaf petioles. The ddPCR assay was 100% accurate in detecting S. citri-infected versus –negative trees and exhibited reproducible quantitation of the pathogen without need of an external standard curve regardless of low target concentration in field samples.