Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a serious disease of susceptible citrus in Florida and other citrus-growing areas of the world. The effect of leaf preconditioning as a route for entry of the bacteria is poorly characterized. A series of experiments were designed to investigate the effects of wind and rain (to simulate a storm), high humidity (>90%) and mild abrasion with sand (to simulate wind-blown sand and debris) in predisposing citrus foliage to infection with Xcc. Exposure of leaves of Swingle citrumelo seedlings to wind and rain [16 m sec(-1) (58 km h(-1), 36 mph) and 235 mm h(-1) (9.25 ins h(-1)), respectively] for 15 or 30 min caused significant injury and disease incidence was twice as high and severity was ten times greater than on leaves on seedlings of unexposed control plants. The points of attachment of the lamina to the petioles were particularly susceptible to wind-induced injury with up to 25% showing symptoms compared to 0% for the unexposed control. Over 80% of injured leaves had lesions associated with the site of injury. There was little or no effect of humidity >90% for 1.5 or 2.5 h on disease incidence or severity compared to the unexposed control. Mild leaf abrasion of grapefruit seedlings with sand increased incidence and severity of disease two-fold. Ways to reduce leaf injury by minimizing wind speed in orchards may contribute to reducing canker incidence and severity.

Citrus canker (Xanthomonas citri subsp. citri, Xcc) is one of the most serious diseases citrus in Florida, and elsewhere in the world. The disease causes yield loss and some fresh fruit trade restrictions may apply. Cultural management techniques such as windbreaks may work by not only reducing wind speed, but also reducing the period of exposure of susceptible foliage or fruit to those wind speeds that support infection from incoming inoculum. To investigate the effect of exposure period to inoculum of Xcc, seedlings of canker-susceptible Swingle citrumelo were exposed to sprayed inoculum for increasing periods at different wind speeds. The incidence and severity of citrus canker was assessed. In three experiments the incidence and severity of citrus canker most often increased with longer periods of exposure to inoculum, especially so at wind speeds of ≥16 m/s compared to wind speeds of ≤5 m/s (wind speed also increased disease incidence and severity). Regression analysis demonstrated relationships between period of exposure to inoculum and the percent infected leaves per plant, the number of lesions per plant, the number of lesions per infected leaf, and for the percent of infected leaves with lesions on the petioles at wind speeds of ≥16 m/s (R2 = 0.16–0.72). Due to the effect of inoculum exposure period and wind speed, attempts should be made to minimize exposure of canker-susceptible citrus when wind speed is highest and inoculum is available. Windbreaks should help minimize periods of exposure to splashed inoculum in high winds.

Citrus canker (Xanthomonas citri subsp. citri (Xcc)) can cause yield loss and trade restrictions. The pathogen is dispersed in rain splash and spread is promoted by wind. The goal of this study was to gain some insight into the properties of short‐distance splash dispersal of Xcc from ∼1·5 m‐tall cankered grapefruit canopies in turbulent wind, common during rainstorms in Florida. Turbulent wind up to 19·9 m s−1 was tested in five experiments. Bacteria flux density (BFD, bacteria cm−2 min−1) was quantified at heights of 30, 70, 110, 130 and 180 cm above ground, and at four horizontal points (17, 51, 85 and 119 cm) at each height across the direction of the wind 1 m downwind. BFD varied among experiments, but the lowest BFDs were consistently detected at the greatest sample height. Despite differences between experiments, the relationship between log BFD and sample height was consistently described by a linear function (P = 0·06–<0·0001, R2 = 0·75–>0·99). The BFD collected at the horizontal points across the wind path was variable. BFDs collected were sometimes significantly different, but no relationship was discernible. Stronger, turbulent wind resulted in greater BFD, with a linear function describing the relationship between log BFD and wind speed (P = 0·2–0·02, R2 = 0·94–0·96). Multiple regression analysis demonstrated predictability of the proportion of total bacteria collected (F = 141, P < 0·0001, d.f. = 3, R2 = 0·53).

Citrus canker (Xanthomonas citri subsp. citri, Xcc) is now considered endemic in Florida and continues to spread. Various surfaces, including plant material, personnel and equipment can become contaminated. Decontamination is practiced in both disease-endemic and disease-free areas to reduce the risk of bacterial spread by man or machinery. We used grapefruit leaf surfaces to explore the efficacy of a commonly used personnel sprayer system applying a quaternary amine decontaminant. In three experiments, plants in flush (leaves 3/4 expanded) were inoculated (similar to 10(5) CFU ml(-1)). Immediately after inoculation, plants were passed through the sprayers 0, 1, 2, 3, or 6 times. Leaves were sampled at 0.5, 10 and 20 min after decontamination and tested for viable Xcc by dilution plating. There was a large and rapid decline in the quantity of live bacteria with one pass through the decontamination sprayer (> 80% decrease in CFU ml(-1)), and multiple sprays (2-6) resulted in up to 100% mortality of surface Xcc. Presumably more thorough coverage with multiple sprays killed remnant bacteria, although the first spray invariably caused the highest proportion of population mortality. The effect of the decontaminant spray was immediate (within 0.5 min only 3-11% of surface bacteria survived, and by 20 min < 1-3% survived). Based on these results, use of a personnel sprayer with a quaternary amine compound is highly effective for reducing surface inoculum. A single spray kills a high proportion of the population, but multiple sprays increase mortality of Xcc. All the Xcc-inoculated plants subsequently developed symptoms of citrus canker. No significant difference in incidence or severity of grapefruit leaf infection was detected among decontamination treatments or compared to the untreated control. This finding indicates that infection occurred at, or very soon after, inoculation, and that Xcc was in protected sites inside the leaf before exposure to the decontaminant spray. Published by Elsevier Ltd.

Xanthomonas citri subsp. citri (Xcc), which causes citrus canker, is a major pathogen of grapefruit and other canker-susceptible citrus species and cultivars grown in Florida and elsewhere. It is dispersed by rain splash, and wind promotes the dispersal of the pathogen. The aim of this study was to explore the relationships between wind speed and the number of bacteria dispersed downwind in rain splash at different heights and distances. Wind (up to 20 m sec(-1)) was generated using a fan, and rain simulated using sprayer nozzles. A negative exponential function described the relationship between height and the bacteria flux density (BFD, bacteria cm(-2) min(-1)), with a rapid decline in mean BFD of Xcc collected at greater heights (P = < 0.0001-0.3838, R(2)= 0.56 - 1.00). A power function described the relationship between distance and BFD of Xcc collected at most heights and wind speeds (P=0.049 - < 0.0001, R(2) = 0.78-1.00). A multiple regression analysis based on wind speed, height and distance suggested predictability of the proportion of the total BFD of Xcc collected downwind of the simulated wind-rain system (F=49, P < 0.0001, R(2)=0.46). Crop management options to reduce sources of inoculum and the wind speed in orchards are discussed.

Citrus canker (caused by the bacterial pathogen Xanthomonas citri subsp. citri, Xcc) can cause severe damage to citrus. It is endemic in Florida, and occurs in other citrus growing regions. The bacterium is dispersed predominantly in rain splash. To simulate dispersal in splash, and to investigate the effect of wind speed on infection, young plants of Swingle citrumelo were exposed to sprayed inoculum at different wind speeds. Wind was generated using an axial fan, and a pressurized sprayer delivered the inoculum spray. In the five experiments, higher wind speeds (>10 m s−1) consistently resulted in higher incidence and severity of citrus canker developing. By 15 ms−1, there was a dramatic increase in disease. Visible injury to leaves of Swingle citrumelo due to wind was evident at wind speeds ≥ 13 m s−1. The relationship between wind speed and disease, and wind speed and injury was described by a logistic model. More disease was associated with visible injury as the wind speed increased, and disease not associated with visible injury also increased with wind speed. The petiole-leaflet junction was more often infected at higher wind speeds (≥17 m s−1). The concentration of the Xcc inoculum increased the incidence and severity of citrus canker in all experiments. Reducing wind speed in citrus groves with the aid of wind breaks may contribute to a reduction in the severity of an epidemic by reducing dispersal and infection events.

The epidemic of citrus canker (Xanthomonas citri subsp. citri) in Florida continues to expand since termination of the eradication program in 2006. Storms are known to be associated with disease spread, but little information exists on the interaction of fundamental physical and biological processes involved in dispersal of this bacterium. To investigate the role of wind speed in dispersal, wind/rain events were simulated using a fan to generate wind up to 19 m·s-1 and spray nozzles to simulate rain. Funnels at ground level and panels at 1.3 m height and distances up to 5 m downwind collected wind-driven splash. Greater wind speeds consistently dispersed more bacteria, measured by concentration (colony forming units [CFU] ml-1) or number sampled (bacteria flux density [BFD] = bacteria cm-2 min-1), from the canopy in the splash. The CFU ml-1 of X. citri subsp. citri collected by panels 1 m downwind at the highest wind speed was up to 41-fold greater than that collected at the lowest wind speed. BFD at the highest wind speed was up to 884-fold higher than that collected at the lowest wind speed. Both panels at distances >1 m and funnels at distances >0 m collected many-fold more X. citri subsp. citri at higher wind speeds compared to no wind (up to 1.4 × 103-fold greater CFU ml-1 and 1.8 × 105-fold the BFD). The resulting relationship between wind speed up to 19 m·s-1 and the mean CFU ml-1 collected by panel collectors downwind was linear and highly significant. Likewise, the mean CFU ml-1 collected from the funnel collectors had a linear relationship with wind speed. The relationship between wind speed and BFD collected by panels was generally similar to that described for CFU ml-1 of X. citri subsp. citri collected. However, BFD collected by funnels was too inconsistent to determine a meaningful relationship with increasing wind speed. The quantity of bacteria collected by panels declined with distance, and the relationship was described by an inverse power model (R2 = 0.94 to 1.00). At higher wind speeds, more bacteria were dispersed to all distances. Windborne inoculum in splash in subtropical wet environments is likely to be epidemiologically significant, as both rain intensity and high wind speed can interact to provide conditions conducive for dispersing large quantities of bacteria from canker-infected citrus trees. Disease and crop management aimed at reducing sources of inoculum and wind speeds in a grove should help minimize disease spread by windborne inoculum.

Citrus canker assessment data were used to investigate effects of using the Horsfall-Barratt (H-B) scale to estimate disease compared to direct estimation to the nearest percent. Twenty-eight raters assessed each of two-hundred infected leaves (0–38% true diseased area). The data were converted to the H-B scale. Correlation (r) showed that direct estimates had higher inter-rater reliability compared to H-B scaled data (r = 0.75 and 0.71 for direct estimates and H-B scaled data, respectively). Lin’s concordance correlation (LCC, ρ c ) analysis showed individual rater estimates by direct estimation had better agreement with true values compared to H-B scaled data. The direct estimates were more precise compared to H-B scaled data (r = 0.80–0.95 and 0.61–0.90, respectively), but measures of generalised bias or accuracy (C b ) were similar for both methods (0.38–1.00). Cumulative mean disease and cumulative variance of the means were calculated for each rater on a leaf-by-leaf basis. Direct estimates were closer to the true severity 59.5% of the time, and to the cumulative true sample mean 53.7% of the time, and to the cumulative true sample mean variance 63.6% of the time. Estimates of mean severity for each leaf based on estimates by 3, 5, 10, 20 and 28 raters were compared to true disease severity. LCC showed that rater-means based on more raters had better agreement with true values compared to individual estimates, but H-B scale data were less precise, although with means based on ≥ 10 raters, agreement was the same for both assessment methods. Magnitude and dispersion of the variance of the means based on H-B scaled data was greater than that by direct estimates. H-B scaling did not improve reliability, accuracy or precision of the estimate of citrus canker severity compared to direct visual estimation.

Citrus canker (Xanthomonas citri subsp. citri) is destructive in many citrus production regions in tropical and subtropical parts of the world. Assessment of canker symptoms is required for diverse reasons, including monitoring epidemics, evaluating the efficacy of control strategies, and disease response in breeding material. The objectives were to compare the ability of experienced and inexperienced raters at assessing citrus canker, to identify factors that affect the quality of the assessment, to determine common sources of error, and to discern how error is related to actual disease magnitude. Two-hundred digital leaf images (0 to 37% area infected) were assessed once by 28 raters, five of whom were experienced plant pathologists (PPs), and 23 who had no experience in disease severity assessment (NPPs). True disease (lesion number [LN], % necrotic area [%N], and % chlorotic+necrotic area [%CN]) was measured using image analysis on a leaf-by-leaf basis, and each parameter was estimated by the 28 raters. LN was neither severely over- nor underestimated, while %N was greatly overestimated, with a lesser tendency to overestimate %CN over the true severity range of these two symptom types. A linear relationship existed between estimate of the disease and true disease for all measures of severity. Data were heteroscedastic and error was not constant with increasing true disease. Agreement between rater estimates and true disease was measured with Lin's concordance correlation coefficient (p,). LN showed greatest agreement (rho(c) = 0.88 to 0.99), followed by %CN (rho(c) = 0.80 to 0.95) and %N (p, = 0.19 to 0.84). Greater lesion number resulted in overestimation of area infected for both %N and %CN. Overestimation was particularly noticeable at low disease severities. There was a linear relationship between log variance and log true disease for LN (r(2) = 0.71), %N (r(2) = 0.85), and %CN (r2 = 0.88), and raters tended to estimate disease above 10% to the nearest 5 or 10% GLM analysis showed differences between PP and NPP groups in assessing disease. For LN precision of assessment for both groups was similar (r(2) > 0.92 and 0.94, respectively), but for estimates of %N and %CN, the PPs were more precise (%N and %CN, r(2) = 0.61 and 0.73, respectively) compared to NPPs (%N and %CN, r(2) = 0.45 and 0.58, respectively). Absolute error for mean LN was low. The absolute error of %N and %CN showed overestimation to approximately 8% area infected. Above 8%, absolute error increased, but comprised both over- and underestimation. For %N and %CN, relative error was almost exclusively positive and dramatic at severity <8% (up to approximately 600%), but at severity >10% it was relatively small. Error in rater estimates of canker severity is ubiquitous. Understanding these sources of error will aid in the development of both appropriate training and relevant rating aids.

Citrus canker (caused by Xanthomonas citri subsp. citri) is a destructive disease, reducing yield and rendering fruit unfit for fresh sale. Accurate assessment of citrus canker severity and other diseases is needed for several purposes, including monitoring epidemics and evaluation of germplasm. We compared measurements of citrus canker severity (percent area infected) from automated image analysis to visual estimates by raters and true values using images from five leaf samples (65, 123, 50, 50, and 200 leaves; disease severity from 0 to 60%). Severity on leaves was measured by automated image analysis by (i) basing threshold values on a presample of leaves, or (ii) replacing healthy leaf color on a leaf-by-leaf basis before automating image analysis. Samples 1 to 4 were assessed by three trained plant pathologists, and sample 5 was assessed by an additional 25 raters. Healthy leaf area color replacement gave the most consistent agreement with the true severity data. Using color replacement, agreement with true values based on Lin's concordance correlation coefficient (ρc) was 0.93, 0.79, 0.71, 0.85, and 0.89 for each of the samples, respectively. The range and consistency of agreement was generally less good for automated thresholds based on a presample (ρc = 0.35-0.90) or visual raters (ρc = 0.30-0.94). The constituents of agreement (precision and accuracy) showed similar trends. No one rater or method was best for every leaf sample, but replacing healthy color in each leaf with a standard color before automation of image analysis improved agreement, and was relatively quick (20 s per image). The accuracy and precision of automated image analysis of citrus canker severity can be comparable to unaided, direct visual estimation by many raters.

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, Cb = 0.99 to 1.00) compared to visual raters (Cb = 0.85 to 1.00). Lesion number was the most reproducible symptom assessment (Lin's concordance correlation coefficient, ρc, = 0.76 to 0.99), followed by % area necrotic+chlorotic (ρc = 0.85 to 0.97), and finally % area necrotic (ρc = 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 (Cb = 0.93 to 0.99) which was more consistently accurately reproduced by VRs than either % area necrotic (Cb = 0.85 to 0.99) or % area necrotic+chlorotic (Cb = 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.

Citrus canker is a disease of citrus and is caused by the bacterial pathogen Xanthomonas citri subsp. citri. Ways of managing the disease are being sought, and accurate, precise, reproducible disease assessment is needed for monitoring epidemics. The objective of this study was to investigate the characteristics of visual assessment of citrus canker symptoms compared with actual disease measured using image analysis (IA). Images of 210 citrus leaves with a range of incidence and severity of citrus canker were assessed by three plant pathologists (VR1-3) and by IA. The number of lesions (L), % area necrotic (%AN), and % area necrotic+chlorotic (%ANC) were assessed. The best relationships were found between %AN and %ANC (r2 = 0.41 to 0.87), and the worst between L and %AN (r2 = 0.27 to 0.66). Bland-Altman plots showed various sources of rater error in assessments, including under- and over-estimation, proportional error, and heterogeneity of variation dependent on actual disease magnitude. There was a tendency to overestimate area diseased, but not lesion counts, and this tendency was pronounced at lower disease severity, with a leaf having more lesions tending to be assessed as having greater area infected compared with a leaf with fewer lesions but equal actual area infected. The rater estimations of disease were less accurate or precise with increasing actual disease severity as indicated by the fit of a normal probability density function—the incidence of extreme values increases with increasing actual disease. For example, for %ANC the kurtosis of the distribution ranged from 17.92 to 1.18, 0.51, and 0.22 in actual disease category ranges of 0 to 10, 11 to 20, 21 to 30, and 31 to 40% area infected, respectively. The log variance of the estimates plotted against log actual disease for all three raters over two assessment occasions gave a linear relationship for L, %AN, and %ANC (r2 = 0.74, 0.65, and 0.74, respectively). Training should improve the accuracy, precision, and reproducibility of raters, and knowledge of the characteristics of disease assessment should help develop and target the training more appropriately and address specific causes and sources of error.