The Horsfall-Barratt scale and severity estimates of citrus canker

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.     

Reliability and accuracy of visual estimation of phomopsis leaf blight of strawberry

ABSTRACT Six different individuals (raters) assessed the severity of Phomopsis leaf blight on strawberry leaflets in five experimental repetitions over 2 years by making a direct visual estimation of the percentage of diseased area of each leaflet or by using the Horsfall-Barratt (H-B) disease scale. Intra-rater and inter-rater reliability and accuracy were determined, and then the relationship between visually estimated severity values and actual severity values was evaluated. Agreement in estimated disease severity values between assessment times by the same raters (i.e., intra-rater reliability), and agreement in disease severity values among raters at a single assessment time (i.e., inter-rater reliability), were both high, with most correlation coefficients being greater than 0.85. The intra-class correlation for overall agreement among raters ranged from 0.80 to 0.96 for the five repetitions. Based on the concordance coefficient calculated for each rater in each repetition, agreement between estimated and actual severity (i.e., accuracy) was somewhat lower than reliability. The relationship between estimated and actual severity was linear, and there was a slight trend to overestimate disease severity. The H-B scale was not more reliable or accurate than direct estimation of severity, and the linear relationship between estimated and actual severity did not support the principles underling the H-B scale. Both size of leaflets and number of lesions per leaflet slightly affected the error in estimate of disease severity.     

A comparison between visual estimates and image analysis measurements to determine septoria leaf blotch severity in winter wheat

Methods to estimate disease severity vary in accuracy, reliability, ease of use and cost. Severity of septoria leaf blotch (SLB, caused by Zymoseptoria tritici) was estimated by four raters and by image analysis (assumed actual values) on individual leaves of winter wheat in order to explore accuracy and reliability of estimates, and to ascertain whether there were any general characteristics of error. Specifically, the study determined: (i) the accuracy and reliability of visual assessments of SLB over the full range of severity from 0 to 100%; (ii) whether certain 10% ranges in actual disease severity between 0 and 100% were more prone to estimation error compared with others; and (iii) whether leaf position affected accuracy within those ranges. Lin's concordance correlation analysis of all severities (0–100%) demonstrated that all raters had estimates close to the actual values (agreement: ρ_c = 0·92–0·99). However, agreement between actual SLB severities and estimates by raters was less good when compared over short 10% subdivisions within the 0–100% range (ρ_c = ?0·12 to 0·99). Despite common rater imprecision at estimating low and high SLB severities, individual raters differed considerably in their accuracy over the short 10% subdivisions. There was no effect of leaf position on accuracy or precision of severity estimate on separate leaves (L1–L3). Pursuing efforts in understanding error in disease estimation should aid in improving the accuracy of assessments, making visual estimates of disease severity more useful for research and applied purposes.     

In the eye of the beholder: the effect of rater variability and different rating scales on QTL mapping

The agronomic importance of developing durably resistant cultivars has led to substantial research in the field of quantitative disease resistance (QDR) and, in particular, mapping quantitative trait loci (QTL) for disease resistance. The assessment of QDR is typically conducted by visual estimation of disease severity, which raises concern over the accuracy and precision of visual estimates. Although previous studies have examined the factors affecting the accuracy and precision of visual disease assessment in relation to the true value of disease severity, the impact of this variability on the identification of disease resistance QTL has not been assessed. In this study, the effects of rater variability and rating scales on mapping QTL for northern leaf blight resistance in maize were evaluated in a recombinant inbred line population grown under field conditions. The population of 191 lines was evaluated by 22 different raters using a direct percentage estimate, a 0-to-9 ordinal rating scale, or both. It was found that more experienced raters had higher precision and that using a direct percentage estimation of diseased leaf area produced higher precision than using an ordinal scale. QTL mapping was then conducted using the disease estimates from each rater using stepwise general linear model selection (GLM) and inclusive composite interval mapping (ICIM). For GLM, the same QTL were largely found across raters, though some QTL were only identified by a subset of raters. The magnitudes of estimated allele effects at identified QTL varied drastically, sometimes by as much as threefold. ICIM produced highly consistent results across raters and for the different rating scales in identifying the location of QTL. We conclude that, despite variability between raters, the identification of QTL was largely consistent among raters, particularly when using ICIM. However, care should be taken in estimating QTL allele effects, because this was highly variable and rater dependent.     

Genetic diversity of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> strains from Mexico associated with Moko disease

The aim of this study was to develop and validate a standard area diagram set (SADs) to assess the severity of peach rust, caused by Tranzschelia discolor. The proposed SADs includes ten images of leaves with a range of severity (0.1, 0.5, 1.0, 2.0, 5.0, 10, 15, 20, 25 and 30%). The SADs was validated by 14 raters who had no experience in plant disease severity estimation. In the first step of the validation, the raters made severity estimates of 50 leaves with a range of rust severity without using SADs. In the second step, the same raters estimated severity of rust on the same 50 leaves using the SADs to aid estimation. Lin’s concordance correlation analysis showed that both precision and accuracy improved when the raters used the SADs compared to the assessments made without SADs. Accuracy, as measured by the coefficient of bias (C _b ) improved from 0.70 to 0.98, without and with SADs, respectively, and precision measured by the correlation coefficient (r) improved from 0.85 to 0.90, without and with SADs, respectively. Overall agreement, measured by Lin’s concordance correlation coefficient (ρ _c ), improved from 0.59 to 0.88 without and with SADs, respectively. Furthermore, estimates were more reliable when using SADs: the coefficient of determination (R²) was 0.60 without and 0.73 with SADs; and the intra-class correlation coefficient (ρ) was 0.72 without, and 0.86 with SADs. Thus, the use of SADs improved the precision, accuracy and reliability of visual estimates of severity of peach rust.     

Development and validation of standard area diagrams to aid assessment of pecan scab symptoms on fruit

Pecan scab (Fusicladium effusum) causes losses of pecan nutmeat yield and quality in the southeastern United States. Disease assessment relies on visual rating, which can be inaccurate and imprecise, with poor inter‐rater reliability. A standard area diagram (SAD) set for pecan scab on fruit valves was developed. A set of 40 images of diseased fruit valves with known severity was assessed twice by 23 raters. The first assessment was conducted without SADs, and the second assessment was made using the SADs as an aid. SADs improved rater accuracy (correction factor, C_b = 0·86 and 0·97, without and with SADs, respectively) and agreement (Lin’s concordance correlation coefficient, ρ_c = 0·79 and 0·89 without and with SADs, respectively) with true values. SADs improved inter‐rater reliability (intra‐class correlation coefficient, ρ = 0·77 and 0·96 without and with SADs, respectively). The least accurate and precise raters without SADs improved more using SADs compared to the most accurate and precise raters. Experienced raters had significantly higher accuracy and precision compared to inexperienced raters, but only when unaided by the SAD set. There was no significant difference in time to assess images without SADs, but experienced raters using SADs were faster compared to inexperienced raters. There was a slight tendency for faster raters to assess more slowly, and slower raters to assess faster when using SADs. SADs improve rater estimates of pecan scab severity on fruit, and this SAD set should be useful for assessment where greater precision, accuracy and inter‐rater reliability are required.     

Effects of rater bias and assessment method on disease severity estimation with regard to hypothesis testing

The effects of bias (over‐ and underestimates) in estimates of disease severity on hypothesis testing using different assessment methods was explored. Nearest percentage estimates (NPE), the Horsfall–Barratt (H‐B) scale, and two linear category scales (10% increments, with and without additional grades at low severity) were compared using simulation modelling to assess effects of bias. Type I and type II error rates were used to compare two treatment differences. The power of the H‐B scale and the 10% scale were least for correctly testing a hypothesis compared with the other methods, and the effects of rater bias on type II errors were greater over specific severity ranges. Apart from NPEs, the amended 10% category scale was most often superior to other methods at all severities tested for reducing the risk of type II errors. It should thus be a preferred method for raters who must use a category scale for disease assessments. Rater bias and assessment method had little effect on type I error rates. The power of the hypothesis test using unbiased estimates was most often greater compared with biased estimates, regardless of assessment method. An unanticipated observation was the greater impact of rater bias compared with assessment method on type II errors. Knowledge of the effects of rater bias and scale type on hypothesis testing can be used to improve accuracy and reliability of disease severity estimates, and can provide a logical framework for improving aids to estimate severity visually, including standard area diagrams and rater training software.     

Assessing disease severity: accuracy and reliability of rater estimates in relation to number of diagrams in a standard area diagram set

Error in estimates of plant disease severity occur and standard area diagrams (SADs) help improve accuracy and reliability. The effects of diagram number in SADs are unknown. The objective of this study was to compare estimates of pecan scab severity made without SADs, and using three‐, five‐, seven‐ or 10‐diagram SADs. Disease severity was estimated to the nearest percent (NPE), or classified to the closest reference diagram value using a scale. Twelve raters assessed 20 images of scabbed pecan valves with and without the SADs using NPEs and the scale method (values were converted to midpoints prior to analysis). Increases in diagram number using NPEs did not necessarily result in more accurate or reliable estimates. Inter‐rater reliability was positively correlated with number of diagrams using NPEs (r = 0·3288 (P < 0·0001)) or a scale (r = 0·2803 (P < 0·0001)). The least accurate estimates improved the most using SADs with NPEs, but the gain did not relate to number of diagrams; as few as three diagrams reduced error of inaccurate estimates as much as five, seven or 10 diagrams. When used as a scale, only estimates made with 10‐diagram SADs had similar accuracy and inter‐rater reliability to estimates made using SADs with NPEs. Maximum disease severity and the disease severity range for the pathosystem and the SADs are important factors and will probably influence resulting accuracy and reliability. The ramifications of diagram numbers in SADs and assessment methods on accuracy and reliability of disease estimates are discussed.     

Development and validation of a set of standard area diagrams to aid in estimation of spot blotch severity on wheat leaves

This study aimed to develop and validate a standard area diagram (SAD) set to quantify the severity of spot blotch, caused by Bipolaris sorokiniana, on wheat leaves. The proposed SAD set includes images of leaves with 11 distinct disease severities (0·1, 1, 5, 10, 20, 30, 40, 50, 60, 70 and 83%). The SAD set was validated by 12 raters without experience in evaluating plant disease. Lin's concordance correlation analysis of estimated versus actual disease severity (based on image analysis) showed that precision and accuracy improved for all raters using the SAD set in contrast to assessments made without it. The SAD set improved accuracy (coefficient of bias, C_b = 0·88 and 0·99, without and with the SAD set, respectively) and agreement (Lin's concordance correlation coefficient, ρ_c = 0·81 and 0·96 without and with the SAD set, respectively) of the estimates of severity. The severity estimates were also more reliable when using the SAD set (coefficient of determination, R² = 0·76 unaided and R² = 0·92 with the SAD set, and intra‐class correlation ρ = 0·79 without the SAD set and ρ = 0·95 using the SAD set). The SAD set proposed in this study will improve the accuracy and reliability of estimates of spot blotch severity on wheat leaves.     

Development and validation of a standard area diagram set to assess blast severity on wheat leaves

This study aimed to develop and validate a standard area diagram set (SADS) to quantify the severity of blast, caused by Pyricularia oryzae, on wheat leaves. The SADs has ten levels: 0.1, 1, 5, 10, 22, 32, 42, 52, 62 and 72 % blast severity. To validate the SADs, 12 inexperienced raters estimated disease severity on 50 images of leaves from cultivars BR-18 (susceptible) and BRS-229 (partially resistant). Blast severity was first estimated without the use of the SADs on 50 leaves with a range of blast severity. The same raters evaluated the same 50 leaves using the SADs as an aid. The SADs improved accuracy (coefficient of bias, C _b ?=?0.88 and 0.99, without and with SADs, respectively) and agreement (Lin’s concordance correlation coefficient, ρ _c ?=?0.84 and 0.96 without and with SADs, respectively) of the estimates of severity. The absolute error was (-) 52 % without the SADs and (-) 24 % when using SADs as an aid. Severity estimates were more reliable when using SADs (R²?=?0.87 unaided and R²?=?0.92 with SAD). The SADs proposed in this study will improve accuracy and reliability of estimates of blast severity on wheat leaves.     

Development and validation of a set of standard area diagrams to estimate severity of potato early blight

Standard area diagrams (SADs) to assess the severity of potato early blight (Alternaria grandis) on leaves of potato (Solanum tuberosum L.) were developed and validated. The proposed SADs include images of leaves with 10 distinct disease severities (0.1, 1, 3, 5, 10, 20, 40, 60, 80 and 100 %). The SADs were validated by 12 raters who had no previous experience in evaluating plant disease. Lin’s concordance correlation analysis of estimated vs. actual disease severity (based on image analysis) showed that precision and accuracy improved for most raters using the SADs, compared to assessments made without the SADs. The SADs improved accuracy (coefficient of bias, C _b ?=?0.97 and 0.99, without and with SADs, respectively) and agreement (Lin’s concordance correlation coefficient, ρ _c ?=?0.91 and 0.98 without and with SADs, respectively) of the estimates of severity. Severity estimates were more reliable when using SADs (coefficient of determination, R ² ?=?0.80 unaided and R ² ?=?0.95 with SADs, and the intra-class correlation ρ?=?0.86 without SADs and ρ?=?0.97 using the SADs). The SADs improved raters’ ability to accurately, precisely and reliably estimate potato early blight severity, and as such can be used to assess severity for several purposes, including breeding for resistance, fungicide screening, and pathotype characterization.     

Development and implementation of cost-effective strategies to manage brown rot of peach trees in Imathia,Greece

Development of cost-effective strategies to manage brown rot caused by Monilinia laxa of peach implies a better understanding of the susceptibility of different cultivars and pre-harvest contamination. This study investigated the susceptibility of 24 peach and nectarine cultivars to shoot blight caused by Monilinia spp. and found various levels of susceptibility, with the nectarine cultivar Tasty Free scored as the most susceptible. Studies on the the existence and detection of latent infections by Monilinia spp. in three peach (‘A37’, ‘Andross’, and ‘E-45’) and three nectarine (‘Venus’, ‘Fantasia’, and ‘Tasty Free’) cultivars were also conducted. The results showed that latent infections were detected only in nectarine cultivars when fruit were collected on 23 May and 22 June. In contrast, nectarine fruit collected on 7 June and all peach cultivars tested had no detectable latent infection. This study also indicated that the fungicide thiophanate methyl applied at the pit hardening stage reduced significantly the percentage of latent infection and subsequently preharvest fruit rots. Finally, a disease forecast model to predict blossom blight, caused by M. laxa, was evaluated in the Prefecture of Imathia, Greece. Trees, sprayed according to the model predictions, showed a statistically lower percentage of blighted shoots than those of unsprayed trees.     

Development and evaluation of a standard area diagram set for the severity of phomopsis leaf blight on eggplant

A standard area diagram set (SADs) with eight severity values ranging from 0.5 to 32% was evaluated as a tool to improve the accuracy of the estimates of Phomopsis leaf blight severity on eggplant by ten inexperienced raters. A first assessment in a 50-leaf dataset of digital images was performed unaided (UN, no SAD). Two further aided (A) assessments, conducted two (A1) and four weeks after the first (A2) unaided one were performed. The precision of the estimates, as indicated by the Pearson’s correlation coefficient (r), improved significantly when using the aid (A1: r = 0.96 and A2: r = 0.96) compared to unaided (UN: r = 0.79). However, the generalized bias (C _b) was not significantly affected because the unaided estimates were already quite accurate (C _b = 0.93). The overall concordance (ρ _c) was significantly improved due to the large gains in precision when using the SADs. The raters’ estimates were more uniform (ρ _c > 0.9) when using the aid, with progressive gains in the reliability of the estimates among them, indicated by the concordance correlation coefficient statistics. The SADs proposed in this study will be useful in severity estimation during field work involving multiple raters, especially as use of the SADs results in less variable estimates, improving accuracy and reliability among raters.     

Improving sooty blotch and flyspeck severity estimation on apple fruit with the aid of standard area diagrams

Sooty blotch and flyspeck is caused by numerous species of fungi that colonize the surface of apple fruit and thereby lower its market value. Although this disease poses a substantial threat to apple growers’ profitability in some regions, reliable and cost-effective methods for epidemiological and disease control studies have not been validated, nor are they widely available. We modified a standard area diagram to aid sooty blotch and flyspeck severity assessments and quantified its impact on accuracy and precision of visual estimates. Samples of ‘Fuji’ and ‘Mutsu’ fruit were photographed both from the top and laterally. Severity was assessed from a sub-sample of 160 images using image analysis software. Validation of the diagram was performed by eight raters who independently assessed severity in two series of selected images representing the lateral view and the top view, initially unaided and subsequently with the aid of the scale. Severity estimates ranged from 0.4% to 98% (most fruit had <10% severity). Accuracy and precision of the estimates were significantly improved when using the diagrammatic scale; concordance correlation coefficient values increased from 0.81 to 0.95. A strong tendency to underestimate severity for the mid-range to high levels was minimized when using the aid, which also improved reproducibility of the estimates among raters. In addition to strengthening evidence that a standard area diagram can be used reliably in sooty blotch and flyspeck studies, we expanded its application to disease assessment in the peduncle region, which enhances the usefulness of the method for evaluating efficacy of management practices.     

Development and validation of a standard area diagram set to aid assessment of severity of loquat scab on fruit

A standard area diagram set (SAD) to aid visual assessment of loquat scab (caused by Fusicladium eriobotryae) severity on fruit was developed and evaluated for improving accuracy, precision and reliability of visual estimates. The SAD set contains eight black and white diagrams of diseased fruit with severity values from 2 % to 98 %. To evaluate the SADs, a group of 20 raters (comprising 10 ‘experienced’ and 10 ‘inexperienced’ raters) assessed the same set of 50 images three times, the first without SADs and the second and third using the SADs as an aid. Only for the group of inexperienced raters did SADs significantly improve accuracy (bias correction factor, C _b?=?0.93 without SADs and 0.98 with SADs), precision (correlation coefficient, r?=?0.88 without SADs and r?=?0.96 with SADs) and overall agreement (Lin’s concordance correlation coefficient, ρ _c?=?0.82 without SADs and ρc with SADs = 0.95) of the estimates. Accuracy and precision of the estimates by inexperienced raters were significantly higher than those obtained by the experienced raters, especially for the second assessment with SADs. Inter-rater reliability was improved when SADs were used by inexperienced raters, whereas a high degree of intra-rater reliability was obtained by both experienced and inexperienced raters when using SADs. The SADs developed in this study were useful for obtaining more accurate, precise and reliable assessments of loquat scab for inexperienced raters, and should be used as an aid for assessing scab in epidemiological studies or monitoring for decision-making purposes.     

Standard area diagram set for assessment of severity and temporal progress of apple blotch

Apple blotch (Marssonina coronaria) is a foliar disease of increasing importance globally. Methods to quantify the disease and knowledge about epidemiology are required for new studies on the disease. The objectives of this study were to develop a standard area diagram set (SADs) to assess apple blotch severity and to describe the temporal progress of the disease under field conditions on older and younger leaves of two apple cultivars. For the development of SADs, symptomatic leaves were collected and scanned to obtain the actual severity. Based on the pattern of the disease, a SADs was elaborated and validated. Leaf severities were estimated without and with SADs by 12 raters to validate the tool. After validation, the SADs was used to assess apple blotch in older and younger leaves of selected shoots during the vegetative cycle of cvs. Eva and Gala. SADs severities ranged from 0.2 to 96%. Accuracy, precision, and reliability of the estimates were significantly improved when the SADs was used. In the field, the onset of apple blotch was in late spring and greater increases in severity occurred during summer. Rates of disease progress ranged from 0.09 to 0.13. Epidemics were different in older and younger leaves at the end of the apple vegetative cycle for both cultivars tested. Our work provided a tool for apple blotch quantification and described the disease progress curve under subtropical conditions, which can contribute as basis for future studies.

     

The Role of Psychophysics in Phytopathology: The Weber–Fechner Law Revisited

The accuracy and precision of disease severity assessment data might be improved if there was a better understanding of how the laws of psychophysics actually relate to the theory and practice of phytopathometry. In this regard, we utilized a classical method developed in the field of psychophysics (the method of comparison stimuli) to test Horsfall and Barratt’s claim that raters cannot accurately discriminate disease severity levels between 25% and 50% because, according to the Weber–Fechner law, visual acuity is proportional to the logarithm of the intensity of the stimulus. We show for two pathosystems, wheat leaf rust and grapevine downy mildew, that raters can accurately discriminate disease severity levels between 25% and 50%, and that although Weber’s law appears to hold true, Fechner’s law does not. Furthermore, based upon our results, the relationship between actual (true) disease severity (X) and disease severity estimated by raters (Y) is linear, not logarithmic as proposed by Horsfall and Barratt.     

First report of Almond witches’ broom phytoplasma1 (‘Candidatus Phytoplasma phoenicium’) causing a severe disease on nectarine and peach trees in Lebanon

Symptoms of shoot proliferation characteristic of phytoplasma diseases were observed on nectarine (Prunus persica var. nucipersica) and peach (P. persica) trees in the Sarada plain, south of Lebanon. The presence of phytoplasmas in the two orchards visited was confirmed by nested polymerase chain reaction using universal primers. The amplified DNA fragments were cloned and sequenced. Blast analysis of over 1000 nucleotides demonstrated the presence of ‘Candidatus Phytoplasma phoenicium’ which is considered to be the causal agent of Almond witches’ broom. This phytoplasma which belongs to the pigeon pea witches’ broom group (16SrIX) can be devastating since Almond witches’ broom has killed thousands of almond trees in Lebanon and Iran. Previous reports indicated that Almond witches’ broom may be transmitted by grafting to peach and nectarine under experimental conditions. This is the first report of a natural and epidemic spread of ‘Ca. Phytoplasma phoenicium’ in peach and nectarine. Farmers in the region were advised to eradicate the infected trees immediately. Further studies on the epidemiology of ‘Ca. Phytoplasma phoenicium’ and its vector(s) are recommended in order to develop successful eradication or disease management programmes.     

Evaluation of peach and nectarine cultivars in Bulgaria for their resistance to plum pox potyvirus

In spring and summer 1989, it was established that plum pox potyvirus (PPV) was present in certain peach cultivars in Bulgaria. At the same time, we started to investigate the distribution of PPV in naturally infected 4–5 year-old peach and nectarine cultivars and hybrids in order to optimize PPV detection. Over 160 peach and nectarine cultivars and hybrids were evaluated. In about 40% of the genotypes, typical plum pox symptoms were observed. The latter were estimated and divided into groups depending on their susceptibility to PPV. Observations were made on the population density of seven aphid species established in the peach orchards. Five proved to be vectors of the virus. Myzus persicae was the vector that played the main role in spreading the virus on peach.     

Expression of phytoplasma‐induced witches’ broom disease symptoms in acid lime (Citrus aurantifolia) trees is affected by climatic conditions

Witches’ broom disease (WBD), caused by ‘Candidatus Phytoplasma aurantifolia’, is a serious disease of acid lime (Citrus aurantifolia) in Oman and the UAE. However, little is known about the distribution of phytoplasma and the expression of WBD symptoms in different geographical locations. A survey was carried out in 18 districts in Oman and the UAE covering 143 orchards and 5823 acid lime trees. ‘Candidatus Phytoplasma aurantifolia’ was detected in acid lime in all the 18 surveyed districts. However, the development of typical symptoms of WBD was only observed in 12 districts. Districts in which the phytoplasma was present but symptoms were not expressed were located either in desert areas or in areas characterized by semitropical conditions. Phylogenetic analysis of 16 phytoplasma isolates from trees developing WBD symptoms and six phytoplasma isolates from trees with no WBD symptoms showed that all isolates share an identical 16S rRNA sequence, belonging to subgroup II‐B. Quantitative PCR analysis showed that the concentration of phytoplasma is significantly higher (8800–801 000 copies) in leaves developing WBD symptoms compared to 2–268 copies in symptomless leaves from the same trees and 8–874 copies in acid lime trees from areas where disease symptoms were not expressed. The lack of expression of WBD symptoms under certain environmental conditions may suggest that symptom development and phytoplasma are affected by certain unfavourable environmental conditions. These findings could provide a basis for managing WBD through encouraging lime cultivation under climatic conditions less conducive to WBD symptom expression.