Transcriptome profile of pecan scab resistant and susceptible trees from a pecan provenance collection.

Pecan scab is a devastating disease that causes damage to pecan (Carya illinoinensis (Wangenh.) K. Koch) fruit and leaves. The disease is caused by the fungus Venturia effusa (G. Winter) and the main management practice for controlling the disease is by application of fungicides at 2-to-3-week intervals throughout the growing season. Besides disease-related yield loss, application of fungicides can result in considerable cost and increases the likelihood of fungicide resistance developing in the pathogen. Resistant cultivars are available for pecan growers; although, in several cases resistance has been overcome as the pathogen adapts to infect resistant hosts. Despite the importance of host resistance in scab management, there is little information regarding the molecular basis of genetic resistance to pecan scab.The purpose of this study was to elucidate mechanisms of natural pecan scab resistance by analyzing transcripts that are differentially expressed in pecan leaf samples from scab resistant and susceptible trees. The leaf samples were collected from trees in a provenance collection orchard that represents the natural range of pecan in the US and Mexico. Trees in the orchard have been exposed to natural scab infections since planting in 1989, and scab ratings were collected over three seasons. Based on this data, ten susceptible trees and ten resistant trees were selected for analysis. RNA-seq data was collected and analyzed for diseased and non-diseased parts of susceptible trees as well as for resistant trees. A total of 313 genes were found to be differentially expressed when comparing resistant and susceptible trees without disease. For susceptible samples showing scab symptoms, 1,454 genes were identified as differentially expressed compared to non-diseased susceptible samples. Many genes involved in pathogen recognition, defense responses, and signal transduction were up-regulated in diseased samples of susceptible trees, whereas differentially expressed genes in pecan scab resistant samples were generally down-regulated compared to non-diseased susceptible samples.Our results provide the first account of candidate genes involved in resistance/susceptibility to pecan scab under natural conditions in a pecan orchard. This information can be used to aid pecan breeding programs and development of biotechnology-based approaches for generating pecan cultivars with more durable scab resistance.

Open Access and Reproducibility in Plant Pathology Research: Guidelines and Best Practices.

The landscape of scientific publishing is experiencing a transformative shift toward open access, a paradigm that mandates the availability of research outputs such as data, code, materials, and publications. Open access provides increased reproducibility and allows for reuse of these resources. This article provides guidance for best publishing practices of scientific research, data, and associated resources, including code, in The American Phytopathological Society journals. Key areas such as diagnostic assays, experimental design, data sharing, and code deposition are explored in detail. This guidance aligns with that observed by other leading journals. We hope the information assembled in this paper will raise awareness of best practices and enable greater appraisal of the true effects of biological phenomena in plant pathology.

Visual Assessment of Phony Peach Disease: Evaluating Rater Accuracy and Reliability.

Phony peach disease (PPD), found predominantly in central and southern Georgia, is a re-emerging disease caused by Xylella fastidiosa (Xf) subsp. multiplex. Accurate detection and rapid removal of symptomatic trees are crucial to effective disease management. Currently, peach producers rely solely on visual identification of symptoms to confirm PPD, which can be ambiguous if early in development. We compared visual assessment to quantitative PCR (qPCR) for detecting Xf in 'Julyprince' in 2019 and 2020 (JP2019 and JP2020) and in 'Scarletprince' in 2020 (SP2020). With no prior knowledge of qPCR results, all trees in each orchard were assessed by a cohort of five experienced and five inexperienced raters in the morning and afternoon. Visual identification accuracy of PPD was variable, but experienced raters were more accurate when identifying PPD trees. In JP2019, the mean rater accuracy for experienced and inexperienced raters was 0.882 and 0.805, respectively. For JP2020, the mean rater accuracy for experienced and inexperienced raters was 0.914 and 0.816, respectively. For SP2020, the mean rater accuracy for experienced and inexperienced raters was 0.898 and 0.807, respectively. All raters had false positive (FP) and false negative (FN) observations, but experienced raters had significantly lower FN rates compared with the inexperienced group. Almost all raters overestimated the incidence of PPD in the orchards. Reliability of visual assessments was demonstrated as moderate to good, regardless of experience. Further research is needed to develop accurate and reliable methods of detection to aid management of PPD as both FPs and FNs are costly to peach production.

Transfer of generic Escherichia coli and attenuated Salmonella enterica Typhimurium from the soil to the surface of in-shell pecans during harvest.

During harvest pecan nuts are at risk of contamination with foodborne pathogens from extended contact with the ground. The objective of this study was to determine the potential transfer of Escherichia coli and Salmonella from the ground to in-shell pecans during the harvesting process. Plots (2 m2) were sprayed with 1 L of a rifampicin (rif) resistant strain of either E. coli TVS 353 or an attenuated Salmonella Typhimurium inoculum at a low (∼4 log CFU/ml), mid (∼6 log CFU/ml) or high (∼8 log CFU/ml) concentrations. The following day, nuts were mechanically harvested and samples from each plot were collected at 1 min, 4 h, and 24 h. Samples were enumerated for Salmonella and E. coli on tryptic soy agar supplemented with rif. The Salmonella levels in the soil from the inoculated plots were 2.0 ± 0.3, 4.1 ± 0.1, and 6.4 ± 0.2 log CFU/g for the low, mid, and high inocula, respectively. The E. coli levels in the soil from the inoculated plots were 1.5 ± 0.4, 3.7 ± 0.3, and 5.8 ± 0.1 log CFU/g for the low, mid, and high inocula, respectively. There was a significant difference in the average daily rainfall among the three trials. Trial 3 received 23.8 ± 9.2 cm, while trials 1 and 2 received much less (0.1 ± 0.1 0.0 ± 0.0 cm, respectively). Inoculation concentration and trial were significant (P<0.05) factors that influenced the transfer of E. coli and Salmonella to pecans. For the high inoculum treatment, bacterial transfer to pecans ranged from 0.7 ± 0.3 to 4.1 ± 0.2 for E. coli and 1.3 ± 0.7 to 4.3 ± 0.4 log CFU/g for Salmonella. For the medium inoculum treatment, transfer ranged from <0.3 to 1.5 ± 0.1 for E. coli and <0.3 to 1.9 ± 0.2 log CFU/g for Salmonella. For the low treatment, transfer ranged from <0.3 to 0.4 ± 0.2 and <0.3 to 0.5 ± 0.1 log CFU/g for E. coli and Salmonella, respectively. These results show the need for implementing agricultural practices that prevent potential transfer of foodborne pathogens onto the surface of in-shell pecans during harvest.

An updated de novo transcriptome for green ash (Fraxinus pennsylvanica).

De novo transcriptome assembly of next-generation sequencing information has become a powerful tool for the study of non-model species. Transcriptomes generated by this method can have high variability due to endless combinations of user-defined variables and programs available for assembly. Many methods have been developed for evaluating the quality of these assemblies. Here, raw sequencing information for Green ash (Fraxinus pennsylvanica Marshall) that was previously published has been re-evaluated. An updated assembly has been developed by including additional sequencing information not used for the currently accepted transcriptome in combination with more stringent trimming parameters. Input reads were assembled with Trinity and Abyss assembly programs. The resulting Trinity assembly has a 7.3-fold increase in genomic breadth of coverage, a 2.4-fold increase in predicted complete open reading frames, an increased L50 value, and increased Benchmarking Universal Single-Copy Ortholog completeness compared to the earlier published transcriptome. This updated transcriptome can be leveraged to help fight the rapid decline of green ash due to pathogens.

Changes in chemical characteristics and modeling sensory parameters of stored pecan nutmeats.

Pecan is a major specialty crop produced in the United States. Sensory evaluation and chemical analyses of pecan nutmeats are integral components of shelf life and have been employed to investigate changes during storage, but there remains a lack of knowledge regarding storage stability. Specifically, the association between shelf life and chemical characteristics has not been investigated. We aimed to investigate the chemical changes in pecan nuts during a range of storage treatments (temperature, relative humidity, packaging material, and modified atmosphere). The results of the chemical analyses were used to build a volatile compound-based sensory prediction model. The work has utility as a rapid method to measure lipid oxidation in pecan, which is of value to the pecan industry. The research also determined a possible association between pecan nut volatile compounds and sensory attributes of pecans, and their perception by human subjects. Building a sensory-based prediction model would reduce dependency on expensive and time-consuming sensory methods.

Influence of Geographical Orchard Location on the Microbiome from the Progeny of a Pecan Controlled Cross.

Carya&nbsp;illinoinensis (Wangenh.) K.Koch production has expanded beyond the native distribution as the genetic diversity of the species, in part, has allowed the trees to grow under broad geographic and climatic ranges. Research in other plant species has demonstrated that the phytobiome enhances their ability to survive and thrive in specific environments and, conversely, is influenced by the prevailing environment and plant genetics, among other factors. We sought to analyze the microbiota of pecan seedlings from the controlled cross 'Lakota' × 'Oaxaca' that were made in Georgia and Texas, respectively, to determine if the maternal geographical origin influences the microbiome of the resulting progeny. No significant differences in bacterial communities were observed between the seeds obtained from the two different states (p = 0.081). However, seed origin did induce significant differences in leaf fungal composition (p = 0.012). Results suggest that, in addition to some environmental, epigenetics, or host genetic components, ecological processes, such as dispersal mechanisms of the host, differentially impact the pecan microbiome, which may have ramifications for the health of trees grown in different environments. Future studies on the role of the microbiome in plant health and productivity will aid in the development of sustainable agriculture for improved food security.

Prevalence and Distribution of Phony Peach Disease (Caused by Xylella fastidiosa) in the United States.

Peach is an important specialty fruit crop in the United States, and phony peach disease (PPD), caused by Xylella fastidiosa subsp. multiplex, has been a major cause of yield loss since it was first observed in 1885. Under a federal eradication program, surveys of PPD were conducted from 1929 to 1972, when the program was terminated. No surveys have been conducted in approximately 50 years; therefore, the current prevalence of PPD in the United States is unknown, especially in the Southeast, where damage was previously most severe. To ascertain the status of PPD, we surveyed orchards in Alabama, Florida, Georgia, and South Carolina from June to August 2020 and, except for South Carolina and northern Georgia, PPD was prevalent. Trees in 17 orchards were subjected to confirmation of X. fastidiosa using the AmplifyRP XRT+ for X. fastidiosa to corroborate our visual assessments; based on these tests, PPD incidence in the orchards ranged from 0 to 30.5%. Ancillary written surveys of relative PPD presence and prevalence were sent to fruit pathologists from universities in 20 states where PPD was historically reported. Only 35.0% of respondents reported that PPD either currently or recently occurred in their state and, of these, three reported PPD to be of significant concern. The results of the physical and written surveys indicate that PPD remains prevalent mainly in the southeastern region of the United States but, in other states where previously reported, it is either not present or has very low prevalence when compared with historical accounts of the disease.

Effect of relative humidity, storage days, and packaging on pecan kernel texture: Analyses and modeling.

The studies expounding on the effects of storage conditions on texture changes are limited. The researchers have been proposing methods to measure pecan texture instrumentally. But current protocols and/or attributes fail to address huge variability during experimentation. Additionally, there are no predictive models to estimate changes in pecan texture during storage. This study addresses all the above concerns and investigates the effects of different relative humidity (RH, 30-90%) and packaging material (Polyethylene-Nylon [PEN], polypropylene [PP], low density polyethylene [LDPE], and metallic laminates [ML]) on pecan texture, introducing a rift ratio (F/H or fracturability to hardness ratio) to address variability in the data and predictive model to estimate changes in the textural attribute of pecans during storage. The textural analysis was conducted on pecan cores and intact pecans to measure the area under curve, fracturability, hardness, cohesiveness, chewiness, springiness, and rift ratio. It was observed that values for the rift ratio obtained using the intact pecan method had high R2 (0.72) as compared to the rest of the textural attributes. A three-parameter logistic model was employed to predict pecan texture during storage. The pecans stored at 75, 80, and 90% reached the rift ratio (F/H) of 0.5 at approx. 115, 3, and 0.15 days (~ 4 hr), respectively. Similarly, pecans stored in LDPE, PP, and PEN packs at 80% reached rift ratio (F/H) of 0.5 at approx. 26, 57, and 78 days, respectively. The presence of any kind of package delayed fracturability loss by at least eight folds at 80% RH. The pecans stored in ML did not experience a significant change in textural attributes.

Multiple Mutations and Overexpression in the CYP51A and B Genes Lead to Decreased Sensitivity of Venturia effusa to Tebuconazole.

Multiple demethylation-inhibiting (DMI) fungicides are used to control pecan scab, caused by Venturia effusa. To compare the efficacy of various DMI fungicides on V. effusa, field trials were conducted at multiple locations applying fungicides to individual pecan terminals. In vitro assays were conducted to test the sensitivity of V. effusa isolates from multiple locations to various concentrations of tebuconazole. Both studies confirmed high levels of resistance to tebuconazole. To investigate the mechanism of resistance, two copies of the CYP51 gene, CYP51A and CYP51B, of resistant and sensitive isolates were sequenced and scanned for mutations. In the CYP51A gene, mutation at codon 444 (G444D), and in the CYP51B gene, mutations at codon 357 (G357H) and 177 (I77T/I77L) were found in resistant isolates. Expression analysis of CYP51A and CYP51B revealed enhanced expression in the resistant isolates compared to the sensitive isolates. There were 3.0- and 1.9-fold increases in gene expression in the resistant isolates compared to the sensitive isolates for the CYP51A and CYP51B genes, respectively. Therefore, two potential mechanisms-multiple point mutations and gene over expression in the CYP51 gene of V. effusa isolates-were revealed as likely reasons for the observed resistance in isolates of V. effusa to tebuconazole.

Population Genetic Characteristics and Mating Type Frequency of Venturia effusa from Pecan in South America.

Scab, caused by the plant-pathogenic fungus Venturia effusa, is a major disease of pecan in South America, resulting in loss of quantity and quality of nut yield. Characteristics of the populations of V. effusa in South America are unknown. We used microsatellites to describe the genetic diversity and population structure of V. effusa in South America, and determined the mating type status of the pathogen. The four hierarchically sampled orchard populations from Argentina (AR), Brazil (BRC and BRS), and Uruguay (UR) had moderate to high genotypic and gene diversity. There was evidence of population differentiation (Fst = 0.196) but the correlation between geographic distance and genetic distance was not statistically significant. Genetic differentiation was minimal between the UR, BRC, and BRS populations, and these populations were more clearly differentiated from the AR population. The MAT1-1 and MAT1-2 mating types occurred in all four orchards and their frequencies did not deviate from the 1:1 ratio expected under random mating; however, multilocus linkage equilibrium was rejected in three of the four populations. The population genetics of South American populations of V. effusa has many similarities to the population genetics of V. effusa previously described in the United States. Characterizing the populations genetics and reproductive systems of V. effusa are important to establish the evolutionary potential of the pathogen and, thus, its adaptability-and can provide a basis for informed approaches to utilizing available host resistance and determining phytosanitary needs.

Pecan color change during storage: Kinetics and Modeling of the Processes.

Postharvest changes in pecan nutmeat color are affected by many factors, both internal and external. The temperature, relative humidity (RH) of the surrounding environment, and storage time are major factors contributing to color deterioration of the nutmeats. Kinetic models have long been employed to provide insights into the physical and chemical changes in food systems; however, no kinetic model has been developed describing the color changes of pecan nutmeats during storage. The objective of this research was to determine the effect of temperature, RH and storage time on pecan nutmeat color change. Pecan nutmeats of three commercially important cultivars (Stuart, Pawnee and Desirable) were subjected to different temperatures (20, 30 and 40 °C) and RH conditions (30, 50, 75% and 80%) for up to 450 days in simulated storage. The observed color changes of the pecan nutmeats were measured as lightness, chroma and hue (LCh). Additionally, the USDA pecan color rating scale was digitized to encourage its use among researchers. It was observed that the change in hue followed a zero-order decay whereas change in lightness and chroma followed a first-order decay. The value of the reaction constants ranged from 0.010 to 1.315 day-1. An Arrhenius model was used to estimate the activation energy (Ea) corresponding to different storage conditions. The values revealed significant effects of temperature, RH and storage days on color degradation. The breakdown of flavonoids and reaction products from Maillard browning could be responsible for the formation of the reddish-brown color observed in degraded nutmeats. The kinetic parameters and models were used to develop a user-friendly online interface for predicting color change depending on selected parameters, with illustrations of the resulting pecan color (https://tinyurl.com/uspecans). The results of this study will aid pecan growers, processors and researchers to predict and visualize changes in color of pecan nutmeats during storage under various conditions of temperature and RH, and duration of storage. Although the study used cultivars Stuart, Pawnee and Desirable, the results likely have more general applicability to other cultivars too.

Understanding the ramifications of quantitative ordinal scales on accuracy of estimates of disease severity and data analysis in plant pathology.

The severity of plant diseases, traditionally defined as the proportion of the plant tissue exhibiting symptoms, is a key quantitative variable to know for many diseases but is prone to error. Plant pathologists face many situations in which the measurement by nearest percent estimates (NPEs) of disease severity is time-consuming or impractical. Moreover, rater NPEs of disease severity are notoriously variable. Therefore, NPEs of disease may be of questionable value if severity cannot be determined accurately and reliably. In such situations, researchers have often used a quantitative ordinal scale of measurement-often alleging the time saved, and the ease with which the scale can be learned. Because quantitative ordinal disease scales lack the resolution of the 0 to 100% scale, they are inherently less accurate. We contend that scale design and structure have ramifications for the resulting analysis of data from the ordinal scale data. To minimize inaccuracy and ensure that there is equivalent statistical power when using quantitative ordinal scale data, design of the scales can be optimized for use in the discipline of plant pathology. In this review, we focus on the nature of quantitative ordinal scales used in plant disease assessment. Subsequently, their application and effects will be discussed. Finally, we will review how to optimize quantitative ordinal scales design to allow sufficient accuracy of estimation while maximizing power for hypothesis testing.

Novel associations in antibiosis stemming from an insect pupal cell.

The pupal soil cell of the pecan weevil, Curculio caryae (Coleoptera: Curculionidae), was reported previously to exhibit antibiosis to an entomopathogenic fungus, Beauveria bassiana. The objectives of this study were to examine 1) if the antimicrobial effect occurs in other insects that form pupal cells, 2) whether the effect extends to plant pathogenic fungi, and 3) identify the source of antibiosis in pupal soil cells of C. caryae. Antibiosis of pupal cells against B. bassiana was confirmed in-vitro in three additional curculionids, Diaprepes abbreviatus, Conotrachelus nenuphar, and Pissodes nemorensis, all of which had fewer fungal colonies relative to controls. Pupal soil cells were found to suppress phytopathogenic fungi in-vitro, including suppression of Alternaria solani by D. abbreviatus pupal cell, and that of Monilinia fructicola by C. caryae. The detection of antibiosis of soil cells formed by surface-sterilized insects using sterile soil implies the antimicrobial effect stemmed from inside the insect. Further, a novel biotic mechanism was identified: a bacterium related to Serratia nematodiphila was isolated from C. caryae pupal soil cells and was found to be associated with antibiosis. The bacterial cultures with or without autoclave had similar effects but were not as potent as pupal soil cells for suppressing B. bassiana. Also, autoclaved soil cells and autoclaved bacterial culture suppressed M. fructicola but were not as inhibitory as non-autoclaved soil cells. This indicates that antibiosis may be due to bacterial metabolites, although other factors may also be involved. Our findings suggest potential to develop the antibiotic compounds as novel bio-fungicides to control plant diseases.

Effect of Tractor Speed and Spray Application Volume on Severity of Scab and Fruit Weight at Different Heights in the Canopy of Tall Pecan Trees.

Scab (caused by Venturia effusa) is the most important yield-limiting disease of pecan in the southeastern USA. On susceptible cultivars, the disease is managed using fungicides, but spray coverage is an issue in tall trees. In four experiments, we used an air-blast sprayer to compare scab severity on fruit at 5.0 to 15.0 m height in trees receiving the same dose of fungicide at 468, 935, and 1,871 liter/ha at 2.4 and 3.2 km/h (in two additional experiments fungicides were applied at 4.0 km/h at 470 liter/ha, 4.0 km/h at 940 liter/ha and 4.0 km/h at 1,100 liter/ha). An air-blast sprayer was used for the applications, which included typical recommended active ingredients (a.i.). Nozzles were selected to provide similar proportions of spray to the upper and lower canopy. The treatments (or subsets thereof) were repeated in 2015 to 2017 on cv. Schley and in 2017, 2019, and 2020 on cv. Desirable. All treatments reduced scab compared with the control. Overall, there was no consistent difference among the treatments for severity of scab on foliage, immature fruit, or mature fruit at any height in the canopy up to 15.0 m (maximum height sampled). Fungicide applied at 2.4 or 3.2 km/h at 470 liter/ha was as effective at reducing disease as were the higher volumes (sometimes more so). The scab epidemic severity affected control efficacy. Estimated cost and water savings based on faster speed and lower volume were considerable. These preliminary observations indicate no single volume or speed was consistently superior to control scab; this suggests that, in most seasons, low volumes (higher concentration of a.i.) may be similarly efficacious as high volumes (lower concentration of a.i.) for controlling scab in tall pecan trees and offer greater resource use efficiency.

Effect of Tractor Speed and Spray Application Volume on Spray Coverage at Different Heights in the Canopy of Tall Pecan Trees.

Scab (caused by Venturia effusa) is the most important disease of pecan in the southeastern U.S.A. The yield losses in susceptible cultivars, combined with costs of control, amount to tens of millions of dollars annually. It is known that fungicide coverage from air-blast sprayers declines with height in the canopy, and conversely, disease severity increases. But how application volume (liter/ha) and speed (km/h) affect spray coverage at different heights is unknown. Coverage was quantified using Kromekote cards (CTI Paper USA, Sun Prairie, WI) and Vision Pink dye (GarrCo Products, Converse, IN) at heights of 5.0, 7.5, 10.0, 12.5, and 15.0 m in pecan canopies. An orchard air-blast sprayer was operated at 2.4 and 3.2 km/h applying 468, 935, or 1,871 liters/ha. Nozzles were selected to provide proportionally similar volumes to the upper and lower canopy positions at set speeds. Speeds tested did not affect spray coverage consistently. However, greater volumes resulted in significantly greater spray coverage, but most of that increase was at heights ≤12.5 m. Although there were significant differences among volumes applied at 12.5 m, differences were numerically small. Card orientation had a profound effect on spray coverage at heights ≤12.5 m, with most spray being detected on the cards facing horizontally downward, and least on those facing vertically backward. The study demonstrates that higher volumes result in more coverage, but the effect declines rapidly with height. If disease control achieved with 470 liters/ha is no different (or is more efficacious) compared with >470 liters/ha and is the same at higher speeds (3.2 km/h), savings may be possible in terms of operating time and equipment costs.

Mating Type Idiomorphs, Heterothallism, and High Genetic Diversity in Venturia carpophila, Cause of Peach Scab.

Scab (caused by Venturia carpophila) is a major disease affecting peach in the eastern United States. The aims of the study were to characterize the mating-type loci in V. carpophila, determine whether they are in equilibrium, and assess the population genetic diversity and structure of the pathogen. The mating-type gene MAT1-1-1 was identified in isolate JP3-5 in an available genome sequence, and the MAT1-2-1 gene was PCR amplified from isolate PS1-1, thus indicating a heterothallic structure. Mating-type loci structures were consistent with those of other Venturia spp. (V. effusa and V. inaequalis): the mating-type gene is positioned between APN2 encoding a DNA lyase and a gene encoding a Pleckstrin homology domain. Primers designed to each of the mating-type genes and a reference gene TUB2 were used as a multiplex PCR to screen a population (n = 81) of V. carpophila from various locations in the eastern United States. Mating types in five of the nine populations studied were in equilibrium. Among the 81 isolates, there were 69 multilocus genotypes. A population genetic analysis of the populations with >10 individuals (four populations) showed them to be genetically diverse. Linkage disequilibrium was found in five of nine populations with ≥4 isolates. A discriminant analysis of principal components indicated three genetic clusters, although extensive admixture was observed. Mating-type identification in V. carpophila provides a basis for understanding reproductive methods of the pathogen and can be a basis for further studies of the genetics of the peach scab pathogen.

Reproducibility of the Development and Validation Process of Standard Area Diagram by Two Laboratories: An Example Using the Botrytis cinerea/Gerbera jamesonii Pathosystem.

Standard area diagrams (SADs) are plant disease severity assessment aids demonstrated to improve the accuracy and reliability of visual estimates of severity. Knowledge of the sources of variation, including those specific to a lab such as raters, specific procedures followed including instruction, image analysis software, image viewing time, etc., that affect the outcome of development and validation of SADs can help improve standard operating practice of these assessment aids. As reproducibility has not previously been explored in development of SADs, we aimed to explore the overarching question of whether the lab in which the measurement and validation of a SAD was performed affected the outcome of the process. Two different labs (Lab 1 and Lab 2) measured severity on the individual diagrams in a SAD and validated them independently for severity of gray mold (caused by Botrytis cinerea) on Gerbera daisy. Severity measurements of the 30 test images were performed independently at the two labs as well. A different group of 18 raters at each lab assessed the test images first without, and secondly with SADs under independent instruction at both Lab 1 and 2. Results showed that actual severity on the SADs as measured at each lab varied by up to 5.18%. Furthermore, measurement of the test image actual values varied from 0 to up to 24.29%, depending on image. Whereas at Lab 1 an equivalence test indicated no significant improvement in any measure of agreement with use of the SADs, at Lab 2, scale shift, generalized bias, and agreement were significantly improved with use of the SADs (P ≤ 0.05). An analysis of variance indicated differences existed between labs, use of the SADs aid, and the interaction, depending on the agreement statistic. Based on an equivalence test, the interrater reliability was significantly (P ≤ 0.05) improved at both Lab 1 and Lab 2 as a result of using SADs as an aid to severity estimation. Gain in measures of agreement and reliability tended to be greatest for the least able raters at both Lab 1 and Lab 2. Absolute error was reduced at both labs when raters used SADs. The results confirm that SADs are a useful tool, but the results demonstrated that aspects of the development and validation process in different labs may affect the outcome.