Fig 1.
Comparison of qPCR-confirmed Washington navel tree infected with Huanglongbing (HLB) (Left) and Citrus stubborn disease (CSD) (Right). (A) CLas infected tree in suburban southern California; (B) Blotchy mottle leaves and distorted fruits symptoms of HLB. (C) S. citri infected tree (left) next to a healthy tree (right) in a field in central California; (D) Chlorotic leaves with shortened internodes and (E) smaller misshapen fruit, with seasonal stylar-end greening typical of CSD. HLB pictures provided by Magally Luque-Williams, CDFA.
Table 1.
Primer and probe sequences used for detection of “Candidatus Liberibacter asiaticus” and Spiroplasma citri.
Fig 2.
Calibration curve of triplex qPCR assays with tenfold serial dilution of “Candidatus Liberibacter asiaticus” and Spiroplasma citri infected DNA (400 ng to 0.004 ng).
Gene specific targets were RNR (dotted line) and ORF1 (unbroken line) for detection of CLas and S. citri, respectively, and COX (dash line) for citrus DNA.
Fig 3.
Calibration curve of duplex qPCR assays with tenfold serial dilution of “Candidatus Liberibacter asiaticus” and Spiroplasma citri DNA (400 ng to 0.004 ng) performed using RNR (dotted line), ORF1 (unbroken line) and COX (dash line) gene specific primers.
(a) Detection of CLas by duplex qPCR assay using RNR and COX gene; (b) Detection of S. citri by duplex qPCR assay using ORF1, and COX gene for citrus DNA.
Fig 4.
Thermal gradient droplet digital PCR for optimizing annealing temperature.
(A) RNR of “Candidatus Liberibacter asiaticus”; (B) ORF1 of Spiroplasma citri. Eight ddPCR reactions with an annealing temperature gradient ranging from 53°C to 63°C are divided by vertical dotted yellow lines. The pink line is the threshold, above which are positive droplets (blue/Green) and below are negative droplets (gray) without any target DNA.
Fig 5.
Linear regression of the singleplex and duplex ddPCR assays for RNR plasmid DNA and ORF1 plasmid DNA for detection of “Candidatus Liberibacter asiaticus” (CLas) and Spiroplasma citri (S. citri) respectively.
The Pearson correlation coefficient of singleplex RNR plasmid DNA regression curve (y = 0.8521x-11.405) is 1 and ORF1 plasmid DNA (0.7658x + 6.2768) is 1, respectively. Pearson correlation coefficient of duplex RNR plasmid DNA regression curve (0.871x - 4.3816) is 1 and ORF1 plasmid DNA (0.7414x + 28.637) is 0.999, respectively. The inner error bars indicate the Poisson 95% confidence interval (CI) and the outer error bars show the total 95% CI of replicates. (P<0.0001).
Fig 6.
Linear regression of the singleplex and duplex ddPCR assays for detection of “Candidatus Liberibacter asiaticus” (RNR gene) and Spiroplasma citri (ORF1 gene) in dually infected citrus leaf DNA.
The Pearson correlation coefficient of singleplex RNR and ORF 1 DNA regression curve is 0.999 and 1, respectively. Pearson correlation coefficient of duplex RNR and ORF1 DNA regression curve is 1. The inner error bars indicate the Poisson 95% confidence interval (CI) and the outer error bars show the total 95% CI of replicates. (P<0.0001).
Fig 7.
Intra-assay (A) and Inter-assay (B) variation of duplex ddPCR assay for detection of RNR gene of “Candidatus Liberibacter asiaticus” and ORF1 gene of Spiroplasma citri in ten-fold dilutions of dual infected citrus leaf DNA. Bar represents the average of triplicate ddPCR values of each dilutions. CV means coefficient of variation.
Fig 8.
Influence of citrus leaf petiole extract on quantification of “Candidatus Liberibacter asiaticus” and Spiroplasma citri by duplex ddPCR assays for RNR gene and ORF1 gene respectively.
(A) ddPCR reaction mixture was spiked with different quantity of citrus leaf petiole extract and equal amount of citrus leaf DNA. Error bars denote standard error of inhibition between three replicates of each reaction. (B, C) one dimensional plot showing only one of three replications for RNR gene and ORF1 gene with citrus leaf petiole extracts.