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.     

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 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.     

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 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 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.     

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.     

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.

     

A new standard area diagram set for assessment of severity of soybean rust improves accuracy of estimates and optimizes resource use

Soybean rust (SBR), caused by Phakopsora pachyrhizi, is the most important yield-damaging fungal disease of soybean due to severe reduction in healthy leaf area and acceleration of leaf fall. In experimental research, SBR severity is estimated visually aided/trained by a standard area diagram (SAD) developed and validated during the mid-2000s (Old SAD). In this study, we propose a new SAD set for SBR with six true-colour diagrams following linear increments (c. 15% increments) amended with four additional diagrams at low (<10%) severities, totalling 10 diagrams (0.2%, 1%, 3%, 5%, 10%, 25%, 40%, 55%, 70%, and 84%). For evaluation, 37 raters were split into two groups. Each assessed severity in a 50-image sample (0.25%–84%), first unaided and then using either the Old SAD or the New SAD. Accuracy, precision, and reliability of estimates improved significantly relative to unaided estimates only when aided by the New SAD (accuracy >0.95). Low precision (<0.78) and a trend of underestimation with an increase in severity were the main issues with the Old SAD, which did not differ from unaided estimates. Simulation to evaluate the impact of the errors by different methods on hypothesis tests, showed that the new SAD was more powerful for detecting the smallest difference in mean control (e.g., 70% vs. 65% disease reduction) than the Old SAD; the latter required a 2-fold increase in sample size to achieve the same power. There is a need to improve some SADs, taking advantage of new knowledge and technology to increase accuracy of the estimates, and to optimize both resource use efficiency and management decisions.     

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.     

Reliability and accuracy of visual methods to quantify severity of foliar bacterial spot symptoms on peach and nectarine

The objectives of this study were to assess the reliability and accuracy of visual methods used to quantify the severity of bacterial spot (Xanthomonas arboricola pv. pruni) symptoms and evaluate the effects of rater experience on the quality of disease estimates. Three cohorts of raters differing in experience with disease assessment rated three sets of peach or nectarine leaves (n ≥ 103; disease severity levels from 0% to 100%) by direct estimation of percentage leaf area with symptoms. Four of the experienced raters also rated the leaves using a 1–7 interval scale. Actual disease severity on the leaves was obtained with the APS assess image analysis software. Equivalence tests based on a bootstrap analysis were used to compare the rating scale and direct estimation methods, and to evaluate the effects of rater experience, computer training and human instruction on accuracy and reliability of disease estimates. In concordance analysis of continuous variables, with data from scale converted to percentage, the direct estimation method resulted in more accurate and reliable estimates than the interval scale. Analysing the scale data without conversion to percentage improved the concordance statistics for the scale but not sufficiently to match the direct estimation method. Accuracy was affected more by rater experience and intrinsic ability than reliability. Instruction on disease symptoms resulted in the largest improvement in estimates from inexperienced raters. Accurate and reliable direct estimation of bacterial spot severity on peach and nectarine can be made by raters with varying levels of experience provided they receive sufficient instruction.     

Dispersal of conidia of Fusicladium eriobotryae and spatial patterns of scab in loquat orchards in Spain

Dispersal of conidia of Fusicladium eriobotryae, the causal agent of loquat scab, was investigated in two loquat orchards in Spain from 2010 to 2012. A volumetric spore sampler, horizontally and vertically orientated microscope slides, and rain collectors were placed in loquat fields to trap conidia of F. eriobotryae. No conidia were collected in the volumetric sampler, and highly variable numbers of conidia were collected in the rain gatherers. Large numbers of conidia were collected by microscope slides, particularly by those held in a horizontal orientation compared with those held in a vertical orientation. Approximately 90 % of the F. eriobotryae conidia were collected during rainy periods. Based on ROC and Bayesian analysis, using ≥ 0.2 mm rainfall as a cut-off value resulted in a high probability of correctly predicting actual conidial dispersal, and had a low probability of failing to predict actual conidial dispersal. Based on the index of dispersion and the binary power law, the incidence of loquat scab on fruit was highly aggregated in space between and within trees, and aggregation was influenced by disease incidence. Our results demonstrate, for the first time, that F. eriobotryae is dispersed mainly in rain splash. The results will be integrated into a mechanistic, weather-driven, disease prediction model that should help growers to minimize fungicide application for the management of loquat scab.     

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.     

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.     

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.     

Copper resistance in Xanthomonas campestris pv. campestris affecting crucifers in Trinidad

Fifty-six native isolates collected in 12 farming districts of Trinidad and seven reference strains of Xanthomonas campestris pv. campestris were evaluated for resistance to copper in buffered (pH 7.0) and unbuffered (pH 5.6) nutrient agar media. All isolates and reference strains were pathogenic and elicited typical black rot symptoms on a susceptible variety of Brassica olearceae, ‘Copenhagen Market’. Thirty-four and thirty-three native isolates were highly resistant to copper (growth on?≥?200 ppm copper) in buffered and unbuffered media, respectively; however, all the reference strains were highly susceptible to copper. The mean minimum inhibition concentration for the 56 native isolates was 224.6 ppm copper indicating that high levels of copper resistance are present in X. campestris pv. campestris in Trinidad. The association between growth of the 56 isolates and seven reference stains on buffered and unbuffered media was strong (Pearson’s and Spearman’s r?=?0.93; P?<?0.01) suggesting that either medium can be used to evaluate resistance to copper in X. campestris pv. campestris. There was also a strong association between length of time of continuous applications of copper formulations to treat black rot disease and proportion of the native X. campestris pv. campestris with resistance to copper (Pearson’s r?=?0.96; Spearman’s r?=?0.93); however, there was no association between resistance to copper and aggressiveness at 10 days after inoculation.     

Genetic diversity assessed by SSR markers and chemotyping of Fusarium culmorum causal agent of foot and root rot of wheat collected from two different fields in Tunisia

Fusarium culmorum is a major pathogen able to cause foot and root rot and the incitant of Fusarium head blight in wheat in Tunisia. The aims of the present study were to evaluate by PCR the type of mycotoxins produced, to determine the mating type and to analyse the genetic diversity by microsatellite markers of 82?F. culmorum isolates recovered from two separated Tunisian fields. Specific sequences in the Tri6-Tri5 intergenic region, Tri7 and Tri13 were used to identify 3-AcDON- or 15-AcDON-. All studied F. culmorum isolates, were of the 3-AcDON- type. No 15-AcDON- and NIV types were detected in this research. Both mating types MAT1-1 and MAT1-2 were recovered from the two fields in approximately equal proportions. Five polymorphic microsatellite markers were applied to F. culmorum isolates, to determine the genetic variation in and among populations. Sixty-four haplotypes were identified; the analysis of the population structure did not reveal a strong variation between fields. Total gene diversity (H _T ?=?0.505; H _S ?=?0.497) and analysis of molecular variance confirmed that most of the genetic variability was within populations (Φ _ST ?=?0.033; P?<?0.0039). Gene flow (N _m ?=?31.05) indicated little differentiation among populations. Based on these results, the F. culmorum isolates collected from different fields might be part of one large panmictic population and in addition the low linkage disequilibrium values with high genetic variation within populations suggest that the population is recombining sexually.     

Characterisation of a protein from Venturia inaequalis that induces necrosis in Malus carrying the Vm resistance gene

Apple scab (black spot) is caused by the fungus, Venturia inaequalis. Race 1 isolates of this fungus are avirulent on Malus hosts carrying the resistance gene V_m. Detached leaves from a V_m host (resistant, differential host 5) and ‘Royal Gala’ (susceptible, host 1) were inoculated with a conidial suspension of V. inaequalis. In the resistant reaction, a hypersensitive response (HR), characterised by necrosis and the accumulation of autofluorescent materials in epidermal and mesophyll cells, was observed at the site of fungal penetration. No HR was observed in the susceptible host. V. inaequalis grown in vitro produced an elicitor that induced necrosis, similar to the HR, when infiltrated into leaves of the resistant V_m host. No response, however, was observed in the susceptible host. The elicitor was proteinaceous and a fraction with elicitor activity was isolated using ultra-filtration, acetone precipitation and ion-exchange chromatography. The elicitor activity was resistant to boiling but it was abolished by digestion with proteinase K. The protein fraction contained three major proteins all with low isoelectric points (pI 3·0–4·5). The fraction also elicited necrosis in the differential host 4, but not in any of the other resistant hosts tested, including differential hosts 2, 3, and 6. Therefore, the fraction may contain elicitors with more than one host specificity.     

Real-time PCR as a promising tool to monitor growth of Venturia spp. in scab-susceptible and -resistant apple leaves

Apple scab, the most important disease of apple worldwide, is caused by Venturia inaequalis. Currently, evaluation of fungal pathogenicity and host resistance is based on a symptomatic disease rating. However, this method does not provide an accurate measurement of the degree of infection and cannot detect early fungal development in symptomless leaves. In this study, a Venturia-specific real-time PCR assay was developed using primers designed around the specific internal transcribed spacer 2 (ITS2) region of the 5.8S rRNA gene. Using SYBR? Green I technology, the assay can accurately quantify Venturia DNA over a concentration range of at least five orders of magnitude. Detection sensitivities were in the order of 100?fg. The method was used to quantify Venturia genomic DNA levels in leaves of three apple cultivars with different levels and types of scab resistance and artificially infected with V. inaequalis. The assay clearly discriminated between Venturia levels in monogenic resistant (‘Topaz’), polygenic resistant (‘Discovery’), and susceptible (‘Golden Delicious’) cultivars, and proved especially useful to quantify pathogen levels during the initial latent stage of infection. The real-time PCR data of ‘Golden Delicious’ were consistent with the observed evolution of the degree of sporulation during a time-course experiment. Although measurements were influenced by the presence of co-extracted PCR-inhibitors, the impact of these compounds was independent of the apple cultivar or the initial amount of fungal DNA present. In conclusion, real-time PCR amplification of the ITS2-5.8S rDNA of Venturia spp. is a faster, more objective and more sensitive method to monitor fungal growth and to evaluate host resistance than phenotypic disease rating scores.     

Elimination of common scab sensitive progeny from a potato breeding population using thaxtomin A as a selective agent

Common scab is one of the most important soil‐borne diseases of potato and is difficult to control. Selection of potato breeding lines for resistance to common scab is also cumbersome due to environmental factors influencing symptom development and an erratic spatial distribution of the scab pathogens (Streptomyces spp.) in the field. The bacterial phytotoxin thaxtomin A, which causes scab symptoms, can be used to screen large numbers of potato seedlings for tolerance in vitro, but few studies have investigated whether the results correspond to resistance to common scab observed in the field. In this study, 120 F1 potato progeny from a single cross were screened in vitro by exposing the seedlings to thaxtomin A added to the culture medium. Eighteen genotypes were selected based on high sensitivity or tolerance using shoot growth as the criterion, multiplied in vitro, and tested for resistance to common scab caused by S. turgidiscabies and S. scabies in a glasshouse and in three different fields. Evaluation of ca. 6500 tubers showed that the 18 potato genotypes differed in scab indices and disease severity (P < 0·0001). The relative shoot height in vitro (thaxtomin A used at 0·5 μg mL^?1) and the scab index in the field showed significant correlation (r_s = ?0·463, P = 0·0528, n = 18), also consistent with the results obtained under controlled conditions in the glasshouse. Hence, the in vitro bioassay may be used to discard scab‐susceptible genotypes and elevate the overall levels of common scab resistance in the potato breeding populations.