Visual Rating and the Use of Image Analysis for Assessing Different Symptoms of Citrus Canker on Grapefruit Leaves.

Citrus canker is caused by the bacterial pathogen Xanthomonas axonopodis pv. citri and infects several citrus species in wet tropical and subtropical citrus growing regions. Accurate, precise, and reproducible disease assessment is needed for monitoring epidemics and disease response in breeding material. The objective of this study was to assess reproducibility of image analysis (IA) for measuring severity of canker symptoms and to compare this to visual assessments made by three visual raters (VR1-3) for various symptom types (lesion numbers, % area necrotic, and % area necrotic+chlorotic), and to assess inter- and intra-VR reproducibility. Digital images of 210 citrus leaves with a range of symptom severity were assessed on two separate occasions. IA was more precise than VRs for all symptom types (inter-assessment correlation coefficients, r, for lesion numbers by IA = 0.99, by VRs = 0.89 to 0.94; for %, r for % area necrotic+chlorotic for IA = 0.97 and for VRs = 0.86 to 0.89; and r for % area necrotic for IA = 0.96 and for VRs = 0.74 to 0.85). Accuracy based on Lin's concordance coefficient also followed a similar pattern, with IA being most consistently accurate for all symptom types (bias correction factor, C = 0.99 to 1.00) compared to visual raters (C = 0.85 to 1.00). Lesion number was the most reproducible symptom assessment (Lin's concordance correlation coefficient, ρ, = 0.76 to 0.99), followed by % area necrotic+chlorotic (ρ = 0.85 to 0.97), and finally % area necrotic (ρ = 0.72 to 0.96). Based on the "true" value provided by IA, precision among VRs was reasonable for number of lesions per leaf (r = 0.88 to 0.94), slightly less precision for % area necrotic+chlorotic (r = 0.87 to 0.92), and poorest precision for % area necrotic (r = 0.77 to 0.83). Loss in accuracy was less, but showed a similar trend with counts of lesion numbers (C = 0.93 to 0.99) which was more consistently accurately reproduced by VRs than either % area necrotic (C = 0.85 to 0.99) or % area necrotic+chlorotic (C = 0.91 to 1.00). Thus, visual raters suffered losses in both precision and accuracy, with loss in precision estimating % area necrotic being the greatest. Indeed, only for % area necrotic was there a significant effect of rater (a two-way random effects model ANOVA returned a P < 0.001 and 0.016 for rater in assessments 1 and 2, respectively). VRs showed a marked preference for clustering of % area severity estimates, especially at severity >20% area (e.g., 25, 30, 35, 40, etc.), yet VRs were prepared to estimate disease of <1% area, and at 1% increments up to 20%. There was a linear relationship between actual disease (IA assessments) and VRs. IA appears to provide a highly reproducible way to assess canker-infected leaves for disease, but symptom characters (symptom heterogeneity, coalescence of lesions) could lead to discrepancies in results, and full automation of the system remains to be tested.