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.

Virulence Comparison of a Comprehensive Panel of Xylella fastidiosa Pierce's Disease Isolates from California.

Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, has been found in all major grape-growing regions in California, U.S.A. Large collections of X. fastidiosa isolates are available from these areas, which enable comparative studies of pathogen genetic traits and virulence. Owing to the significant resource requirements for experiments with X. fastidiosa in grapevine, however, most studies use only a single isolate to evaluate disease, and it is not clear how much variability between isolates impacts disease development in experimental or natural settings. In this study, a comprehensive panel of X. fastidiosa isolates from all California grape-growing regions was tested for virulence in susceptible grapevine and in the model host plant, tobacco. Seventy-one isolates were tested, 29 in both grapevine and tobacco. The results of this study highlight the inherent variability of inoculation experiments with X. fastidiosa, including variation in disease severity in plants inoculated with a single isolate, and variability between experimental replicates. There were limited differences in virulence between isolates that were consistent across experimental replicates, or across different host plants. This suggests that choice of isolate within the X. fastidiosa subsp. fastidiosa Pierce's disease group may not make any practical difference when testing in susceptible grape varieties, and that pathogen evolution has not significantly changed virulence of Pierce's disease isolates within California. The location of isolation also did not dictate relative disease severity. This information will inform experimental design for future studies of X. fastidiosa in grapevine and provide important context for genomic research.

High Growing Season Temperatures Limit Winter Recovery of Grapevines from Xylella fastidiosa Infection - Implications for Epidemiology in Hot Climates.

Management of widespread plant pathogens is challenging as climatic differences among crop-growing regions may alter key aspects of pathogen spread and disease severity. Xylella fastidiosa is a xylem-limited bacterial pathogen that is transmitted by xylem sap-feeding insects. Geographic distribution of X. fastidiosa is limited by winter climate, and vines infected with X. fastidiosa can recover from infection when held at cold temperatures. California has a long history of research on Pierce's disease and significant geographic and climatic diversity among grape-growing regions. This background in combination with experimental disease studies under controlled temperature conditions can inform risk assessment for X. fastidiosa spread and epidemic severity across different regions and under changing climate conditions. California's grape-growing regions have considerable differences in summer and winter climate. In northern and coastal regions, summers are mild and winters are cool, conditions which favor winter recovery of infected vines. In contrast, in inland and southern areas, summers are hot and winters mild, reducing likelihood of winter recovery. Here, winter recovery of three table grape cultivars (Flame, Scarlet Royal, and Thompson Seedless) and three wine grape cultivars (Sauvignon Blanc, Cabernet Sauvignon, and Zinfandel) were evaluated under temperature conditions representative of the San Joaquin Valley, an area with hot summers and mild winters that has been severely impacted by Pierce's disease and contains a large portion of California grape production. Mechanically inoculated vines were held in the greenhouse under one of three warming treatments to represent different seasonal inoculation dates prior to being moved into a cold chamber. Winter recovery under all treatments was generally limited but with some cultivar variation. Given hot summer temperatures of many grape-growing regions worldwide, as well as increasing global temperatures overall, winter recovery of grapevines should not be considered a key factor limiting X. fastidiosa spread and epidemic severity in the majority of cases.

Development of a PNA-LNA-LAMP Assay to Detect an SNP Associated with QoI Resistance in Erysiphe necator.

The repetitive use of quinone outside inhibitor fungicides (QoIs, strobilurins; Fungicide Resistance Action Committee [FRAC] 11) to manage grape powdery mildew has led to development of resistance in Erysiphe necator. While several point mutations in the mitochondrial cytochrome b gene are associated with resistance to QoI fungicides, the substitution of glycine to alanine at codon 143 (G143A) has been the only mutation observed in QoI-resistant field populations. Allele-specific detection methods such as digital droplet PCR and TaqMan probe-based assays can be used to detect the G143A mutation. In this study, a peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA-LAMP) assay consisting of an A-143 reaction and a G-143 reaction, was designed for rapidly detecting QoI resistance in E. necator. The A-143 reaction amplifies the mutant A-143 allele faster than the wild-type G-143 allele, while the G-143 reaction amplifies the G-143 allele faster than the A-143 allele. Identification of resistant or sensitive E. necator samples was determined by which reaction had the shorter time to amplification. Sixteen single-spore QoI-resistant and -sensitive E. necator isolates were tested using both assays. Assay specificity in distinguishing the single nucleotide polymorphism (SNP) approached 100% when tested using purified DNA of QoI-sensitive and -resistant E. necator isolates. This diagnostic tool was sensitive to one-conidium equivalent of extracted DNA with an R2 value of 0.82 and 0.87 for the G-143 and A-143 reactions, respectively. This diagnostic approach was also evaluated against a TaqMan probe-based assay using 92 E. necator samples collected from vineyards. The PNA-LNA-LAMP assay detected QoI resistance in ≤30 min and showed 100% agreement with the TaqMan probe-based assay (≤1.5 h) for the QoI-sensitive and -resistant isolates. There was 73.3% agreement with the TaqMan probe-based assay when samples had mixed populations with both G-143 and A-143 alleles present. Validation of the PNA-LNA-LAMP assay was conducted in three different laboratories with different equipment. The results showed 94.4% accuracy in one laboratory and 100% accuracy in two other laboratories. The PNA-LNA-LAMP diagnostic tool was faster and required less expensive equipment relative to the previously developed TaqMan probe-based assay, making it accessible to a broader range of diagnostic laboratories for detection of QoI resistance in E. necator. This research demonstrates the utility of the PNA-LANA-LAMP for discriminating SNPs from field samples and its utility for point-of-care monitoring of plant pathogen genotypes.

Fungicide Resistance and Host Influence on Population Structure in Botrytis spp. from Specialty Crops in California.

Botrytis is an important genus of plant pathogens causing pre- and postharvest disease on diverse crops worldwide. This study evaluated Botrytis isolates collected from strawberry, blueberry, and table grape berries in California. Isolates were evaluated for resistance to eight different fungicides, and 60 amplicon markers were sequenced (neutral, species identification, and fungicide resistance associated) distributed across 15 of the 18 B. cinerea chromosomes. Fungicide resistance was common among the populations, with resistance to pyraclostrobin and boscalid being most frequent. Isolates from blueberry had resistance to the least number of fungicides, whereas isolates from strawberry had resistance to the highest number. Host and fungicide resistance-specific population structure explained 12 and 7 to 26%, respectively, of the population variability observed. Fungicide resistance was the major driver for population structure, with select fungicides explaining up to 26% and multiple fungicide resistance explaining 17% of the variability observed. Shared and unique significant single-nucleotide polymorphisms (SNPs) associated with host and fungicide (fluopyram, thiabendazole, pyraclostrobin, and fenhexamid) resistance-associated population structures were identified. Although overlap between host and fungicide resistance SNPs were detected, unique SNPs suggest that both host and fungicide resistance play an important role in Botrytis population structure.

Rotting Grapes Don't Improve with Age: Cluster Rot Disease Complexes, Management, and Future Prospects.

Cluster rots can be devastating to grape production around the world. There are several late-season rots that can affect grape berries, including Botrytis bunch rot, sour rot, black rot, Phomopsis fruit rot, bitter rot, and ripe rot. Tight-clustered varieties such as 'Pinot gris', 'Pinot noir', and 'Vignoles' are particularly susceptible to cluster rots. Symptoms or signs for these rots range from discolored berries or gray-brown sporulation in Botrytis bunch rot to sour rot, which smells distinctly of vinegar due to the presence of acetic acid bacteria. This review discusses the common symptoms and disease cycles of these different cluster rots. It also includes useful updates on disease diagnostics and management practices, including cultural practices in commercial vineyards and future prospects for disease management. By understanding what drives the development of different cluster rots, researchers will be able to identify new avenues for research to control these critical pathogens.

Population Genetics and Fungicide Resistance of Botrytis cinerea on Vitis and Prunus spp. in California.

Botrytis cinerea, the causal agent of gray mold, has high genetic diversity and a broad host range. In Vitis sp. and Prunus spp., B. cinerea causes pre- and postharvest diseases, and fungicides are routinely applied to prevent yield loss. In total, 535 isolates of B. cinerea collected from Vitis sp. and Prunus spp. in 2012, 2016, and 2017 were genotyped using 18 microsatellite markers and the transposable elements (TEs) Boty and Flipper. Only nine of the polymorphic markers and the two TEs were considered informative and retained for the final analyses. Of the 532 isolates, 297 were tested for resistance to seven fungicides representing six Fungicide Resistance Action Committee classes. After clone correction, 295 multilocus genotype groups were retained across the 3 years in 326 individuals, and four genetic subpopulations were detected. High levels of clonality were observed across the dataset. Significant pairwise differentiation was detected among years, locations, and TE composition. However, most of the diversity observed was within a subpopulation and not among subpopulations. No genetic differentiation was detected among resistant and sensitive isolates for individual fungicide classes. When resistance to the total number of fungicides was compared, regardless of the fungicide class, significant differentiation was detected among isolates that are resistant to two fungicide classes and those resistant to three or four fungicide groups. Fungicide resistance frequencies were stable for most chemistries evaluated with the exception of fluopyram, which increased from 2012 to 2016/2017.

Characterization, Virulence, Epidemiology, and Management of Anthracnose in Celery.

Leaf curling and petiole twisting of celery (Apium graveolens) were observed in several commercial fields in five Michigan counties in 2010 through 2012, causing significant crop damage and loss. Prior to this time, the pathogen Colletotrichum acutatum species complex had not been previously associated with celery in Michigan. In this study, the pathogen's genotype and phenotype were characterized, the influence of environmental conditions determined, and fungicides tested. Pathogen identification was based on conidial morphology and molecular identification using species-specific primers. Intersimple-sequence repeat (ISSR) banding patterns were similar between C. acutatum isolates from celery (n = 51) and blueberry (n = 1) but different from C. dematium and C. gloeosporioides. Four ISSR primers resulted in 4% polymorphism when tested on isolates from celery. Pathogenicity and virulence of C. acutatum sensu lato isolated from celery (n = 81), tomato (n = 2), and blueberry (n = 1) were evaluated in greenhouse experiments, which revealed differences in virulence among isolates but no significant differences specific to collection year, county, or field. In dew chambers and growth chambers, high temperatures (≥25°C) or long leaf wetness duration (>24 h) increased disease incidence. Twelve fungicides were tested in field studies over two growing seasons to determine their efficacy against celery anthracnose. The fungicides azoxystrobin, pyraclostrobin, mancozeb, and chlorothalonil reduced disease by 27 to 50% compared with the untreated control when disease pressure was moderate.

A New Method for Fractionation and Characterization of Polyphenols and Tannins from Grapevine Leaf Tissue.

Plants accumulate different types of phenolic material in their tissue as a response to biotic as well as abiotic stress. Monomeric polyphenols and smaller oligomers can serve as protection against ultraviolet radiation or prevent oxidative tissue damage, while larger molecules such as tannins can be the plant's reaction to an infection or physical damage. Therefore, characterization, profiling, and quantification of diverse phenolics can provide valuable information about the plant and the stress status at any given time. A method was developed that allows the extraction of polyphenols and tannins from leaf tissue, followed by fractionation and quantification. Extraction was performed with liquid nitrogen and 30% acetate-buffered ethanol. The method was tested with four cultivars under varying extraction conditions (solvent strength and temperature) and showed great improvements of the chromatography that would otherwise be impacted by tannins. The separation of tannins from smaller polyphenols was achieved by bovine serum albumin precipitation and resuspension in a urea-triethanolamine buffer. Tannins were reacted with ferric chloride and analyzed spectrophotometrically. Monomeric non-protein-precipitable polyphenols were then analyzed via HPLC-DAD from the supernatant of the precipitation sample. This way, a more complete spectrum of compounds can be analyzed from the same plant tissue extract. With the fractionation suggested here, hydroxycinnamic acids and flavan-3-ols can be separated and quantified with good accuracy and precision. Possible applications include the assessment of plant stress and response monitoring using the total concentrations of polyphenols and tannins, as well as the ratios between those compound classes.

A contiguous de novo genome assembly of sugar beet EL10 (Beta vulgaris L.).

A contiguous assembly of the inbred 'EL10' sugar beet (Beta vulgaris ssp. vulgaris) genome was constructed using PacBio long-read sequencing, BioNano optical mapping, Hi-C scaffolding, and Illumina short-read error correction. The EL10.1 assembly was 540 Mb, of which 96.2% was contained in nine chromosome-sized pseudomolecules with lengths from 52 to 65 Mb, and 31 contigs with a median size of 282 kb that remained unassembled. Gene annotation incorporating RNA-seq data and curated sequences via the MAKER annotation pipeline generated 24,255 gene models. Results indicated that the EL10.1 genome assembly is a contiguous genome assembly highly congruent with the published sugar beet reference genome. Gross duplicate gene analyses of EL10.1 revealed little large-scale intra-genome duplication. Reduced gene copy number for well-annotated gene families relative to other core eudicots was observed, especially for transcription factors. Variation in genome size in B. vulgaris was investigated by flow cytometry among 50 individuals producing estimates from 633 to 875 Mb/1C. Read-depth mapping with short-read whole-genome sequences from other sugar beet germplasm suggested that relatively few regions of the sugar beet genome appeared associated with high-copy number variation.

Using Cameras for Precise Measurement of Two-Dimensional Plant Features: CASS.

Images are used frequently in plant phenotyping to capture measurements. This chapter offers a repeatable method for capturing two-dimensional measurements of plant parts in field or laboratory settings using a variety of camera styles (cellular phone, DSLR), with the addition of a printed calibration pattern. The method is based on calibrating the camera using information available from the EXIF tags from the image, as well as visual information from the pattern. Code is provided to implement the method, as well as a dataset for testing. We include steps to verify protocol correctness by imaging an artifact. The use of this protocol for two-dimensional plant phenotyping will allow data capture from different cameras and environments, with comparison on the same physical scale. We abbreviate this method as CASS, CAmera aS Scanner.

Identification of SNPs associated with magnesium and sodium uptake and the effect of their accumulation on micro and macro nutrient levels in Vitis vinifera.

Macro and micro nutrient accumulation affects all stages of plant growth and development. When nutrient deficiencies or excesses occur, normal plant growth is altered resulting in symptoms such as leaf chlorosis, plant stunting or death. In grapes, few genomic regions associated with nutrient accumulation or deficiencies have been identified. Our study evaluated micro and macro nutrient concentrations in Vitis vinifera L. to identify associated SNPs using an association approach with genotype by sequencing data. Nutrient concentrations and foliar symptoms (leaf chlorosis and stunting) were compared among 249 F1 Vitis vinifera individuals in 2015 and 2016. Foliar symptoms were consistent (≥90%) between years and correlated with changes in nutrient concentrations of magnesium (r = 0.65 and r = 0.38 in 2015 and 2016, respectively), aluminum (r = 0.24 and r = 0.49), iron (r = 0.21 and r = 0.49), and sodium (r = 0.32 and r = 0.21). Single nucleotide polymorphisms associated with symptoms, sodium, and magnesium were detected on each chromosome with the exception of 5, 7 and 17 depending on the trait and genome used for analyses explaining up to 40% of the observed variation. Symptoms and magnesium concentration were primarily associated with SNPs on chromosome 3, while SNPs associated with increased sodium content were primarily found on chromosomes 11 and 18. Mean concentrations for each nutrient varied between years in the population between symptomatic and asymptomatic plants, but relative relationships were mostly consistent. These data suggest a complex relationship among foliar symptoms and micro and macro nutrients accumulating in grapevines.

Population Genetic Analyses of Botrytis cinerea Isolates From Michigan Vineyards Using a High-Throughput Marker System Approach.

As sequencing costs continue to decrease, new tools are being developed for assessing pathogen diversity and population structure. Traditional marker types, such as microsatellites, are often more cost effective than single-nucleotide polymorphism (SNP) panels when working with small numbers of individuals, but may not allow for fine scale evaluation of low or moderate structure in populations. Botrytis cinerea is a necrotrophic plant pathogen with high genetic variability that can infect more than 200 plant species worldwide. A panel of 52 amplicons were sequenced for 82 isolates collected from four Michigan vineyards representing 2 years of collection and varying fungicide resistance. A panel of nine microsatellite markers previously described was also tested across 74 isolates from the same population. A microsatellite and SNP marker analysis of B. cinerea populations was performed to assess the genetic diversity and population structure of Michigan vineyards, and the results from both marker types were compared. Both methods were able to detect population structure associated with resistance to the individual fungicides thiabendazole and boscalid, and multiple fungicide resistance (MFR). Microsatellites were also able to differentiate population structure associated with another fungicide, fluopyram, while SNPs were able to additionally differentiate structure based on year. For both methods, AMOVA results were similar, with microsatellite results explaining a smaller portion of the variation compared with the SNP results. The SNP-based markers presented here were able to successfully differentiate population structure similar to microsatellite results. These SNP markers represent new tools to discriminate B. cinerea isolates within closely related populations using multiple targeted sequences.

Discovery of the REN11 Locus From Vitis aestivalis for Stable Resistance to Grapevine Powdery Mildew in a Family Segregating for Several Unstable and Tissue-Specific Quantitative Resistance Loci.

Race-specific resistance loci, whether having qualitative or quantitative effects, present plant-breeding challenges for phenotypic selection and deciding which loci to select or stack with other resistance loci for improved durability. Previously, resistance to grapevine powdery mildew (GPM, caused by Erysiphe necator) was predicted to be conferred by at least three race-specific loci in the mapping family B37-28 × C56-11 segregating for GPM resistance from Vitis aestivalis. In this study, 9 years of vineyard GPM disease severity ratings plus a greenhouse and laboratory assays were genetically mapped, using a rhAmpSeq core genome marker platform with 2,000 local haplotype markers. A new qualitative resistance locus, named REN11, on the chromosome (Chr) 15 was found to be effective in nearly all (11 of 12) vineyard environments on leaves, rachis, berries, and most of the time (7 of 12) stems. REN11 was independently validated in a pseudo-testcross with the grandparent source of resistance, "Tamiami." Five other loci significantly predicted GPM severity on leaves in only one or two environments, which could indicate race-specific resistance or their roles in different timepoints in epidemic progress. Loci on Chr 8 and 9 reproducibly predicted disease severity on stems but not on other tissues and had additive effects with REN11 on the stems. The rhAmpSeq local haplotype sequences published in this study for REN11 and Chr 8 and 9 stem quantitative trait locus (QTL) can be used directly for marker-assisted selection or converted to SNP assays. In screening for REN11 in a diversity panel of 20,651 vines representing the diversity of Vitis, this rhAmpSeq haplotype had a false positive rate of 0.034% or less. The effects of the other foliar resistance loci detected in this study seem too unstable for genetic improvement regardless of quantitative effect size, whether due to race specificity or other environmental variables.

Identification of Vitis Cultivars, Rootstocks, and Species Expressing Resistance to a Planococcus Mealybug.

Mealybugs cause economic loss to vineyards through physical damage, fouling fruit and leaves with honeydew, and the transmission of viruses. Planococcus ficus is one of several mealybug species in vineyards, and one that causes economic damage over a relatively large global range. To develop novel management tools, host resistance to P. ficus, which has not previously been identified for any grape cultivars, was studied. Ten grape lines (species, cultivars, and rootstocks) were evaluated for P. ficus resistance across two separate potted plant assays. Significant differences were detected among cultivars and rootstocks in the recorded number of P. ficus juveniles, adults, and egg sacs. Cabernet Sauvignon and Chardonnay were two of the most favorable grape cultivars for mealybug population growth, whereas rootstocks IAC 572, 10-17A, and RS-3 all demonstrated some level of resistance. Southern fire ant (Solenopsis xyloni) was positively associated with mealybug populations, but did not have a negative effect on the observed presence of other arthropod species including potential predators.

Genetic Diversity, Population Structure, and Heritability of Fruit Traits in Capsicum annuum.

Cultivated pepper (Capsicum annuum) is a phenotypically diverse species grown throughout the world. Wild and landrace peppers are typically small-fruited and pungent, but contain many important traits such as insect and disease resistance. Cultivated peppers vary dramatically in size, shape, pungency, and color, and often lack resistance traits. Fruit characteristics (e.g. shape and pericarp thickness) are major determinants for cultivar selection, and their association with disease susceptibility can reduce breeding efficacy. This study evaluated a diverse collection of peppers for mature fruit phenotypic traits, correlation among fruit traits and Phytophthora fruit rot resistance, genetic diversity, population structure, and trait broad sense heritability. Significant differences within all fruit phenotype categories were detected among pepper lines. Fruit from Europe had the thickest pericarp, and fruit from Ecuador had the thinnest. For fruit shape index, fruit from Africa had the highest index, while fruit from Europe had the lowest. Five genetic clusters were detected in the pepper population and were significantly associated with fruit thickness, end shape, and fruit shape index. The genetic differentiation between clusters ranged from little to very great differentiation when grouped by the predefined categories. Broad sense heritability for fruit traits ranged from 0.56 (shoulder height) to 0.98 (pericarp thickness). When correlations among fruit phenotypes and fruit disease were evaluated, fruit shape index was negatively correlated with pericarp thickness, and positively correlated with fruit perimeter. Pepper fruit pericarp, perimeter, and width had a slight positive correlation with Phytophthora fruit rot, whereas fruit shape index had a slight negative correlation.

Genetic diversity, population structure, and resistance to Phytophthora capsici of a worldwide collection of eggplant germplasm.

Eggplant (Solanum melongena L.) is an important solanaceous crop with high phenotypic diversity and moderate genotypic diversity. Ninety-nine genotypes of eggplant germplasm (species (S. melongena, S. incanum, S. linnaeanum and S. gilo), landraces and heirloom cultivars) from 32 countries and five continents were evaluated for genetic diversity, population structure, fruit shape, and disease resistance to Phytophthora fruit rot. Fruits from each line were measured for fruit shape and evaluated for resistance to two Phytophthora capsici isolates seven days post inoculation. Only one accession (PI 413784) was completely resistant to both isolates evaluated. Partial resistance to Phytophthora fruit rot was found in accessions from all four eggplant species evaluated in this study. Genetic diversity and population structure were assessed using 22 polymorphic simple sequence repeats (SSRs). The polymorphism information content (PIC) for the population was moderate (0.49) in the population. Genetic analyses using the program STRUCTURE indicated the existence of four genetic clusters within the eggplant collection. Population structure was detected when eggplant lines were grouped by species, continent of origin, country of origin, fruit shape and disease resistance.