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.

The Nuances of Plant Disease Severity Estimation Using Quantitative Ordinal Scales-Lessons Learned over Four Decades.

We revisit the foundations of the Horsfall-Barratt (HB) scale, a widely cited and applied plant disease visual assessment tool introduced in 1945, a full 37 years prior to T. T. Hebert's 1982 critique that raised concerns regarding the scale's rationale, particularly its reliance on the Weber-Fechner law and visual perception assumptions. Although use of the HB scale and similar ordinal scales persists, comprehensive studies have revealed that direct visual estimation using percentage scales often proves more accurate and reliable. Challenges remain, such as biases due to estimator subjectivity and the potential for misclassification. The logarithmic assumptions of the HB scale have been debunked, and the importance of choosing appropriate interval sizes and numbers of classes in developing ordinal scales is emphasized. Analyzing ordinal scale data appropriately is crucial, and recent advances offer promising methods that reduce type II error rates. The closely related disease severity index is noted to have its shortcomings and potential for misuse. The letter underscores the need for continuous refinement and critical evaluation of disease assessment methodologies.

From Detection to Protection: The Role of Optical Sensors, Robots, and Artificial Intelligence in Modern Plant Disease Management.

In the past decade, there has been a recognized need for innovative methods to monitor and manage plant diseases aiming to meet the precision demands of modern agriculture. Over the last 15 years, significant advances in detection, monitoring, and management of plant diseases have been made, largely propelled by cutting-edge technologies. Recent advances in precision agriculture have been driven by sophisticated tools such as sensors, artificial intelligence, microsensor networks, and autonomous vehicles. These technologies have enabled the development of novel cropping systems, allowing for targeted management of crops, contrasting with traditional, homogeneous treatment of large crop areas. Research in this field is usually highly collaborative, and interdisciplinary endeavor. It brings together experts from diverse fields such as plant pathology, computer science, statistics, engineering, and agronomy. Despite the progress, translating the advancements in the precision of decision-making or automation into agricultural practice remains a challenge. Enhancing the accuracy and timeliness of disease detection continues to be a priority, with data-driven artificial intelligence systems poised to play a pivotal role. This perspective addresses critical questions and challenges faced in the implementation of digital technologies for plant disease management. It underscores the urgency of integrating innovative technological advances with traditional integrated pest management (IPM). It highlights unresolved issues regarding the establishment of control thresholds for site-specific treatments and the necessary alignment of digital technology use with regulatory frameworks. Importantly, the paper calls for intensified research efforts, widespread knowledge dissemination, and education to optimize the application of digital tools for plant disease management.

Key Challenges in Plant Pathology in the Next Decade.

Plant diseases significantly impact food security and food safety. It was estimated that food production needs to increase by 50% to feed the projected 9.3 billion people by 2050. Yet, plant pathogens and pests are documented to cause up to 40% yield losses in major crops, including maize, rice, and wheat, resulting in annual worldwide economic losses of approximately US$220 billion. Yield losses due to plant diseases and pests are estimated to be 21.5% (10.1 to 28.1%) in wheat, 30.3% (24.6 to 40.9%) in rice, and 22.6% (19.5 to 41.4%) in maize. In March 2023, The American Phytopathological Society (APS) conducted a survey to identify and rank key challenges in plant pathology in the next decade. Phytopathology subsequently invited papers that address those key challenges in plant pathology, and these were published as a special issue. The key challenges identified include climate change effect on the disease triangle and outbreaks, plant disease resistance mechanisms and its applications, and specific diseases including those caused by Candidatus Liberibacter spp. and Xylella fastidiosa. Additionally, disease detection, natural and man-made disasters, and plant disease control strategies were explored in issue articles. Finally, aspects of open access and how to publish articles to maximize the Findability, Accessibility, Interoperability, and Reuse of digital assets in plant pathology were described. Only by identifying the challenges and tracking progress in developing solutions for them will we be able to resolve the issues in plant pathology and ultimately ensure plant health, food security, and food safety.

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.

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.

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.

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.

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 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.

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.

Chromosome-Level Reference Genome of Venturia effusa, Causative Agent of Pecan Scab.

Pecan scab, caused by Venturia effusa, is a devastating disease of pecan (Carya illinoinensis), which results in economic losses on susceptible cultivars throughout the southeastern United States. To enhance our understanding of pathogenicity in V. effusa, we have generated a complete telomere-to-telomere reference genome of V. effusa isolate FRT5LL7-Albino. By combining Illumina MiSeq and Oxford Nanopore MinION data, we assembled a 45.2-Mb genome represented by 20 chromosomes and containing 10,820 putative genes, of which 7,619 have at least one functional annotation. The likely causative mutation of the albino phenotype was identified as a single base insertion and a resulting frameshift in the gene encoding the polyketide synthase ALM1. This genome represents the first full chromosome-level assembly of any Venturia sp.

A Comparison of Ground-Based Air-Blast Sprayer and Aircraft Application of Fungicides to Manage Scab in Tall Pecan Trees.

Pecan scab (caused by Venturia effusa) is a destructive disease of pecan in the southeastern United States. Susceptible cultivars must be sprayed with fungicide every 10 to 21 days to ensure yield and kernel quality. Fungicide is most often applied using large orchard air-blast sprayers. Pecan trees grow tall, and air-blast sprays result in a gradient in spray deposition and consequently of scab. Aerial fungicide application is also practiced. Disease distribution and spray deposition of the two methods have not been compared but will provide information aiding decisions on spray application methods. We compared air-blast, aerial, and air-blast + aerial applications for efficacy controlling scab at five heights in the canopy of 25-m cultivar Schley pecan trees. There was a negative relationship between scab severity and height in control trees, a positive linear relationship with height in air-blast treated trees, and a generally negative linear relationship between scab severity and height in aerially treated trees. Air-blast + aerial treatments resulted in low severity of scab at all heights. Spray deposition on water-sensitive cards indicated a declining gradient with height using an air-blast sprayer, whereas aerial applications resulted in a low deposition at all sample heights. Air-blast sprays tended to result in less good control at heights >12.5 m, and aerially treated trees at ≤7.5 m. The results provide insight into the efficacy and advantages of these methods for applying fungicide to control scab in tall pecan trees; further research is needed to better understand the impact of frequency and timing of these two methods.

The Effect of Tractor Speed and Canopy Position on Fungicide Spray Deposition and Peach Scab Incidence and Severity.

Peach scab, caused by Venturia carpophila, is a damaging disease of peach in the southeastern United States. Thus, fungicides are applied to reduce peach scab. Tractor speed was investigated as a variable affecting spray deposition and disease control in relation to volume applied. In experiments in 2015 and 2016, trees were sprayed with fungicide to control scab at petal fall to 1% shuck split and at shuck split to 10% shuck off. Speeds were 3.2, 4.8, and 6.4 kph resulting in 1,403, 935, and 701 liters/ha, respectively, with the dose of active ingredient (a.i.) per ha kept constant. Deposition declined for all speeds with later spray dates. There was a negative linear relationship between tractor speed and spray coverage on three of four dates the experiment was repeated. Tractor speed (different volumes, equal doses) affected peach scab. In 2015 and 2016, mean incidence at 3.2, 4.8, and 6.4 kph was 68.6, 59.2, and 38.3%, and 64.2, 53.0, and 40.4% of fruit scabbed, respectively. Effect of speed on lesion number per fruit depended on year: in 2015, lesions per fruit were reduced at 6.4 kph compared with 3.2 and 4.8 kph but were not different in 2016. Control trees had fewer lesions per fruit high in the canopy, but there was little effect of sample height in fungicide-treated trees. Concentration of a.i. in lower volumes applied at higher speed may provide some benefit in reducing incidence of peach scab, but there appeared to be less effect on severity.

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.

Novel Primers and Sampling for PCR Detection of Xylella fastidiosa in Peach.

Epidemics of phony peach disease (PPD), caused by Xylella fastidiosa, are of increasing concern to peach (Prunus persica) producers in the southeastern United States. Primers suitable for both conventional PCR (cPCR) and quantitative PCR (qPCR), along with optimal tissue and sampling time, are needed for comparative and reliable detection of X. fastidiosa. In this study, we developed and assessed novel primers for X. fastidiosa and for peach and compared detection of X. fastidiosa in four peach tissue types sampled at three time points using both cPCR and qPCR. Primer C06Xf-bamA was extensively tested for reliable detection of X. fastidiosa due to the more consistent intensity of the cPCR products and the marginally lower average quantification cycle (Cq) values of the qPCR products, compared with the other primers screened. Among the four peach tissue types tested, only root samples demonstrated reliable and consistent detection of X. fastidiosa; stem, petiole, and leaf samples, regardless of source trees, primers used, sampling times, or PCR methods (cPCR or qPCR), were unreliable for detection, due to insufficient quantity of DNA of X. fastidiosa in these samples based on the relative quantification assay. The Cq means and ratios were compared and statistically analyzed, to ascertain effects of source tree, tissue type, sampling time, and primer. Differences in detection sensitivity and the Cq means among sampled trees, sampling times, tested primers, and tissues (except root) were not significant or were inconsistent precluding further exploitation. In summary, these novel primers are a useful resource for detecting X. fastidiosa, and based on our results, root is the only tissue type reliable for year-round detection of X. fastidiosa in peach. Further research on potential utilization of above-ground tissues for PCR detection of X. fastidiosa are discussed.

Disease Incidence-Severity Relationships on Leaflets, Leaves, and Fruit in the Pecan-Venturia effusa Pathosystem.

The most destructive disease of pecan in the southeastern United States is scab, caused by Venturia effusa. Incidence (I)-severity (S) relationships have not previously been characterized in this pathosystem, but incidence measures can save time and should have higher accuracy compared with estimates of severity. Ten scab-susceptible cultivars and seedling trees were assessed for I and S of scab on fruit (1,972 trees) and foliage (compound leaves and leaflets, 1,129 trees) between 2010 and 2014. Samples were assessed on a tree basis, and sample size ranged from 10 to 100 specimens per tree. The range in mean I and S was different depending on the organ (fruit I = 0 to 100%, S = 0 to 100%; compound leaves and leaflets, I = 0 to 100%, S = 0-10.1%, respectively). However, mean I could be 100% at a mean S < 2.0% for fruit, compound leaves, and leaflets. Both I and S data were transformed by complementary log-log prior to linear regression analysis. A linear regression model described the relationship between transformed I and S per tree for fruit (P ≤ 0.0001, R2 = 0.61), compound leaves (P ≤ 0.0001, R2 = 0.82), and leaflets (P ≤ 0.0001, R2 = 0.91) for all cultivars. The regression analysis showed significant effects of cultivar and year on the relationship between I and S; therefore, separate analyses were performed for each cultivar and year. Back-transformed predicted severity values showed that the differences among cultivars and years were negligible at severity <80%, and were generally numerically small at severity >80%. The observation that low severity persists until a high incidence of scab is achieved may limit the ability of incidence data to clearly differentiate treatment effects, even when based on the CLL transformation. But if found to be effective, and if used, it would reduce the labor requirements and result in more accurate data being obtained, as incidence estimates do not tend to suffer from the same subjective biases as do visual estimates of severity.

Fine-Scale Population Genetic Structure and Within-Tree Distribution of Mating Types of Venturia effusa, Cause of Pecan Scab in the United States.

Scab (caused by Venturia effusa) is the major disease of pecan in the southeastern United States. There is no information available on the fine-scale population genetic diversity or the occurrence of clonal types at small spatial scales that provides insight into inoculum sources and dispersal mechanisms, and potential opportunity for sexual reproduction. To investigate fine-scale genetic diversity, four trees of cultivar Wichita (populations) were sampled hierarchically: within each tree canopy, four approximately evenly spaced terminals (subpopulations) were selected and up to six leaflets (sub-subpopulations) were sampled from different compound leaves on each terminal. All lesions (n = 1 to 8) on each leaflet were sampled. The isolates were screened against a panel of 29 informative microsatellite markers and the resulting multilocus genotypes (MLG) subject to analysis. Mating type was also determined for each isolate. Of 335 isolates, there were 165 MLG (clonal fraction 49.3%). Nei's unbiased measure of genetic diversity for the clone-corrected data were moderate to high (0.507). An analysis of molecular variance demonstrated differentiation (P = 0.001) between populations on leaflets within individual terminals and between terminals within trees in the tree canopies, with 93.8% of variance explained among isolates within leaflet populations. Other analyses (minimum-spanning network, Bayesian, and discriminant analysis of principal components) all indicated little affinity of isolate for source population. Of the 335 isolates, most unique MLG were found at the stratum of the individual leaflets (n = 242), with similar total numbers of unique MLG observed at the strata of the terminal (n = 170), tree (n = 166), and orchard (n = 165). Thus, the vast majority of shared clones existed on individual leaflets on a terminal at the scale of 10s of centimeters or less, indicating a notable component of short-distance dispersal. There was significant linkage disequilibrium (P < 0.001), and an analysis of Psex showed that where there were multiple encounters of an MLG, they were most probably the result of asexual reproduction (P < 0.05) but there was no evidence that asexual reproduction was involved in single or first encounters of an MLG (P > 0.05). Overall, the MAT1-1-1 and MAT1-2-1 idiomorphs were at equilibrium (73:92) and in most populations, subpopulations, and sub-subpopulations. Both mating types were frequently observed on the same leaflet. The results provide novel information on the characteristics of populations of V. effusa at fine spatial scales, and provide insights into the dispersal of the organism within and between trees. The proximity of both mating idiomorphs on single leaflets is further evidence of opportunity for development of the sexual stage in the field.