Clive H. Bock

Plant Disease Severity Estimated Visually, by Digital Photography and Image Analysis, and by Hyperspectral Imaging

Reliable, precise and accurate estimates of disease severity are important for predicting yield loss, monitoring and forecasting epidemics, for assessing crop germplasm for disease resistance, and for understanding fundamental biological processes including co-evolution. Disease assessments that are inaccurate and/or imprecise might lead to faulty conclusions being drawn from the data, which in turn can lead to incorrect actions being taken in disease management decisions. Plant disease can be quantified in several different ways. This review considers plant disease severity assessment at the scale of individual plant parts or plants, and describes our current understanding of the sources and causes of assessment error, a better understanding of which is required before improvements can be targeted. The review also considers how these can be identified using various statistical tools. Indeed, great strides have been made in the last thirty years in identifying the sources of assessment error inherent to visual rating, and this review highlights ways that assessment errors can be reduced—particularly by training raters or using assessment aids. Lesion number in relation to area infected is known to influence accuracy and precision of visual estimates—the greater the number of lesions for a given area infected results in more overestimation. Furthermore, there is a widespread tendency to overestimate disease severity at low severities (<10%). Both interrater and intrarater reliability can be variable, particularly if training or rating aids are not used. During the last eighty years acceptable accuracy and precision of visual disease assessments have often been achieved using disease scales, particularly because of the time they allegedly save, and the ease with which they can be learned, but recent work suggests there can be some disadvantages to their use. This review considers new technologies that offer opportunity to assess disease with greater objectivity (reliability, precision, and accuracy). One of these, visible light photography and digital image analysis has been increasingly used over the last thirty years, as software has become more sophisticated and user-friendly. Indeed, some studies have produced very accurate estimates of disease using image analysis. In contrast, hyperspectral imagery is relatively recent and has not been widely applied in plant pathology. Nonetheless, it offers interesting and potentially discerning opportunities to assess disease. As plant disease assessment becomes better understood, it is against the backdrop of concepts of reliability, precision and accuracy (and agreement) in plant pathology and measurement science. This review briefly describes these concepts in relation to plant disease assessment. Various advantages and disadvantages of the different approaches to disease assessment are described. For each assessment method some future research priorities are identified that would be of value in better understanding the theory of disease assessment, as it applies to improving and fully realizing the potential of image analysis and hyperspectral imagery.

Effect of Simulated Wind-Driven Rain on Duration and Distance of Dispersal of Xanthomonas axonopodis pv. citri from Canker-Infected Citrus Trees

Dynamics of dispersal of the bacteria that causes citrus canker (Xanthomonas axonopodis pv. citri) were assessed in simulated wind-driven rain splash. The wind/rain-splash events were simulated using electric blowers to generate turbulent wind (15 to 20 m s-1) and sprayer nozzles to produce water droplets entrained in the wind flow. The splash was blown at an inoculum source of canker-infected trees 1 m downwind. The splash downwind of the source of the infected trees was collected by vertical panel samplers and funnel samplers. The duration over which bacteria were dispersed in spray was assessed in continuous wind at intervals from 0 to 52 h after commencing the simulated rain splash event. In one experiment on 11 February 2003, a total of 1.48 × 106 bacteria were collected by panels 1 m downwind from the inoculum source during the first 10 min of dispersal, but the numbers declined to 3.60 × 105 bacteria after 1 h and ranged between 1.42 × 105 and 1.93 × 104 up to 52 h. In a more detailed study (15 July 2003) of dispersal duration over 4 h, the greatest quantity of bacteria collected by panel samplers were dispersed in the first 5-min period (1.01 × 108 bacteria collected). By 10 min after initiation of dispersal, approximately one-third (3.09 × 107 bacteria collected) of the initial number was being dispersed, and by the end of the first hour, only one-tenth (1.31 × 107 bacteria collected) of the initial quantity was dispersed. Funnel samplers placed at ground level under the trees showed a similar trend. The distance to which bacteria were dispersed in wind-blown splash was also tested under simulated conditions: on 18 September 2003, bacteria were collected by panel samplers at all distances sampled (1, 2, 4, 6, 8, 10, and 12 m) with the greatest number of bacteria deposited at 1 m (4.93 × 106 bacteria collected), while 2.22 × 103 bacteria were deposited over a 10-min period 12 m from the inoculum source. Wind speed declined from 19.5 m s-1 upwind of the trees to 2.8 m s-1 1 m downwind, and by 4 m downwind from the inoculum source, movement was similar to the surrounding air. The data on duration and distance of dispersal were best described by power law regression models compared to exponential models. Citrus canker is readily dispersed in wind-driven rain and is dispersed in large quantities immediately after the stimulus occurs, upon which wind-driven splash can disperse inoculum over a prolonged period and over a substantial distance.

Characteristics of the Perception of Different Severity Measures of Citrus Canker and the Relationships Between the Various Symptom Types

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.

Automated Image Analysis of the Severity of Foliar Citrus Canker Symptoms

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 Lins 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.

Wind Speed Effects on the Quantity of Xanthomonas citri subsp. citri Dispersed Downwind from Canopies of Grapefruit Trees Infected with Citrus Canker

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.

Comparison of Assessment of Citrus Canker Foliar Symptoms by Experienced and Inexperienced Raters

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 Lins concordance correlation coefficient (ρc). LN showed greatest agreement (ρc = 0.88 to 0.99), followed by %CN (ρc = 0.80 to 0.95) and %N (ρc = 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 (r2 = 0.71), %N (r2 = 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 (r2 > 0.92 and 0.94, respectively), but for estimates of %N and %CN, the PPs were more precise (%N and %CN, r2 = 0.61 and 0.73, respectively) compared to NPPs (%N and %CN, r2 = 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.

EXACERBATION OF CITRUS CANKER BY CITRUS LEAFMINER PHYLLOCNISTIS CITRELLA IN FLORIDA

ABSTRACT Citrus canker (caused by Xanthomonas citri subsp. citri, Xcc) is an important bacterial disease of citrus that is spread naturally by rain and wind. Feeding damage to citrus leaves by the citrus leafminer (CLM), Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), has been shown to promote infection levels of citrus canker in a number of citrus-growing regions around the world. We conducted 2 studies to document that CLM damage exacerbates canker in Florida citrus. In 1 study, young citrus trees of 5 cultivare commonly grown in Florida were inoculated with a culture of Xcc. Two groups of trees were studied, 1 group with leaves damaged by CLM and 1 group that was treated with a pesticide to prevent CLM damage. Over all 5 cultivars, comparisons between the 2 groups of trees indicated that CLM damage resulted in 6-fold increase in the number of lesions. No difference was found between the 2 groups with respect to numbers of canker lesions on leaves without CLM damage. In the second study, a survey of commercial citrus groves was conducted to investigate incidence of canker on leaves with and without CLM injury. Low percentages of leaves infected by citrus canker were observed during the survey, with a maximum of 15% of leaves infected in 1 grove. However, during late Jul and Aug in some grapefruit and lemon groves, an average of 79% of leaves with canker had lesions directly associated with CLM damage, and an average of 36 more lesions per leaf was present on leaves with CLM damage. Exacerbation of canker by CLM during Jul and Aug coincided with the time of year when environmental conditions are usually optimal for canker in Florida and when population levels of CLM usually are most abundant. Citrus growers managing citrus canker should benefit from controlling CLM during the summer when conditions are favorable for canker infections, particularly in lemons and grapefruits.

What Interval Characteristics Make a Good Categorical Disease Assessment Scale

Plant pathologists most often obtain quantitative information on disease severity using visual assessments. Category scales have been used for assessing plant disease severity in field experiments, epidemiological studies, and for screening germplasm. The most widely used category scale is the Horsfall-Barratt (H-B) scale, but reports show that estimates of disease severity using the H-B scale are less precise compared with nearest percent estimates (NPEs) using the 0 to 100% ratio scale. Few studies have compared different category scales. The objective of this study was to compare NPEs, the H-B midpoint converted data, and four different linear category scales (5 and 10% increments, with and without additional grades at low severity [0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, 20.0…100%, and 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 30.0…100%, respectively]). Results of simulations based on known distributions of disease estimation using the type II error rate (the risk of failing to reject H0 when H0 is false) showed that at disease severity ≤ 5%, a 10% category scale had a greater probability of failing to reject H0 when H0 is false compared with all other methods, while the H-B scale performed least well at 20 to 50% severity. The 5% category scale performed as well as NPEs except when disease severity was ≤ 1%. Both the 5 and 10% category scales with the additional grades included performed as well as NPEs. These results were confirmed with a mixed model analysis and bootstrap analysis of the original rater assessment data. A better knowledge of the advantages and disadvantages of category scale types will provide a basis for plant pathologists and plant breeders seeking to maximize accuracy and reliability of assessments to make an informed decision when choosing a disease assessment method.

The Effect of Horsfall-Barratt Category Size on the Accuracy and Reliability of Estimates of Pecan Scab Severity

Pecan scab (Fusicladium effusum) is a destructive pecan disease. Disease assessments may be made using interval-scale-based methods or estimates of severity to the nearest percent area diseased. To explore the effects of rating method-Horsfall-Barratt (H-B) scale estimates versus nearest percent estimates (NPEs)-on the accuracy and reliability of severity estimates over different actual pecan scab severity ranges on fruit valves, raters assessed two cohorts of images with actual area (0 to 6, 6+ to 25%, and 25+ to 75%) diseased. Mean estimated disease within each actual disease severity range varied substantially. Means estimated by NPE within each actual disease severity range were not necessarily good predictors of the H-B scale estimate at <25% severity. H-B estimates by raters most often placed severity in the wrong category compared with actual disease. Measures of bias, accuracy, precision, and agreement using Lins concordance correlation depended on the range of actual severity, with improvements increasing with actual disease severity category (from 0 to 6 through 25+ to 75%); however, the improvement was unaffected by the H-B assessments. Bootstrap analysis indicated that NPEs provided either equally good or more accurate and precise estimate of disease compared with the H-B scale at severities of 25+ to 75%. Inter-rater reliability using NPEs was greater at 25+ to 75% actual disease severity compared with using the H-B scale. Using NPEs compared with the H-B scale will more often result in more precise and accurate estimates of pecan scab severity, particularly when estimating actual disease severities of 25+ to 75%.

Vertical distribution of scab in large pecan trees.

Pecan scab (Fusicladium effusum) is a destructive disease of pecan in the southeastern United States. This study was conducted to investigate the vertical distribution of scab in tall pecan trees (14 to 16 m tall) in three experiments in 2010 and 2011. Although 2010 had average rainfall, a factor that drives scab epidemics, 2011 was a very dry year with a consequently low scab severity. A total of eight trees were included in each experiment, four were nontreated controls, and four were sprayed using a ground-based air-blast sprayer. Trees were assessed for foliar and fruit scab at 0-5.0, 5.0+-7.5, 7.5+-10.0, 10.0+-12.5, and 12.5+-15.0 m. Mixed model analysis showed main effects of height, fungicide treatment, and height*treatment interactions in all three experiments, although on foliage the effects were less consistent (P value = 0.003-0.8), perhaps due to delayed fungicide applications early in the season. However, fruit of nontreated trees had more severe scab low in the canopy compared to fungicide-treated trees, with a consistent height*treatment interaction (P value = <0.0001-0.04). Most often the severity of scab in the upper canopy was similar in trees on fungicide-treated and nontreated trees, suggesting that fungicide had less impact at heights ≥10.0 m compared to <10.0 m in the canopy. There was a consistent reduction in scab severity on foliage and on immature fruit in August due to fungicide treatment at heights ≤10.0 m. Above 10.0 m, the effect was inconsistent, but late in the season (October), the fungicide-treated trees showed lower scab severity throughout the canopy. A metallic tracer study using cerium (Ce) showed an exponential relationship between quantities of Ce recovered and sample height in the canopy, with the quantity of Ce at ≥10 m being statistically equal to background levels. The relationship between scab severity on fruit and sample height in the canopy of nontreated trees was most often described by a negative linear function, but there was no discernible relationship on fungicide-treated trees, as the severity of scab in the lower canopy was most often similar to that in the upper canopy. Gradients in fungicide coverage and scab severity have ramifications for scab management options and potentially for the development of fungicide resistance in F. effusum.