Performing Department
(N/A)
Non Technical Summary
Dollar spot is one of the most important turf diseases across the world necessitating frequent fungicide applications for control on golf courses. Despite this importance, very little is known about the pathogen population from a genetics level. In this project we aim to understand the genetic diversity of this pathogen across a typical golf course with the hopes of developing a greater understanding of the impact of our current control methods. Furthermore, we will gather an updated view of fungicide insensitivity in the northeast US and investigate the genetics underlying these resistant populations. We will do this by sampling native pathogen populations from golf courses and compare the genetics of these pathogens to previously isolated pathogens from ~14 years ago. By doing so, we can identify how the pathogen population has changed over time, while correlating these changes to historical management practices. These results will be directly informative to golf course superintendents who are managing these diseases, but also provide critical information for scientists investigating fungicide insensitivity and fungal pathogen population biology across many different crops and systems.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The goal of this project is to investigate the population biology of Clarireedia jacksonii, the causative agent of dollar spot, on creeping bentgrass turfgrass within the Northeast United States. This project will test the hypotheses that 1) historical fungicide management result in selection sweeps that shift Clarireedia jacksonii allele frequencies over time 2.) dollar spot epidemics within a single field are more diverse genetically than previously supposed, and 3.) seasonal weather shifts select for distinct dollar spot pathogen sub-populations. These hypotheses will be investigated though comparative genomics of dollar spot isolates either collected from 1.) historically fungicide-insensitive populations on stakeholder fields, 2.) from research fields at high density, and 3.) as bulked-samples across Spring, Summer, and Fall. As such these objectives will be completed via 1.) Assessing dollar spot population diversity and fungicide resistance across select golf courses in the Northeast using Genotyping-by-sequencing and in-vitro fungicide sensitivity, 2.) Assessing dollar spot population genetic diversity within small geographic space and large geographic space using genotyping-by-sequencing and next generationgenomics, and 3.) Determiningif seasonality influence pathogen population structure on golf courses using next generation bulked segregant analysis.
Project Methods
Objectives 1, 2a. and 2b. will be achieved via the following:Objective 1: historical fungicide management results in selection sweeps that shift Clarireedia jacksonii (dollar spot) population allele frequencies over time.i)Resampling of previously described golf course dollar spot populations by Putman et al (2010) across Connecticut and Massachusetts. Golf courses identified with high levels of propiconazole and thiophanate-methyl from previous research conducted by Putman et al. (2010) with be visited in middle summer months of 2024 when dollar spot disease is high (>50 disease probability) according to the Smith-Kerns model (Smith and Kerns et al. 2018). Communication with golf course superintendents will inform exact timing of sampling based on recent fungicide application and disease pressures observed in the field. These golf courses include The Country Club of Farmington (Farmington CT), TPC Boston (Norton, MA), Hartford Golf Club (West Hartford, CT), and Pine Orchard Yacht & Country Club (Branford, CT). A total of 50 isolates from each golf course will be collected from greens, tees, and fairways following the sampling scheme described by Putman et al. (2010). Disease leaf tissue will be incubated on antibiotic-amended potato dextrose agar for one day and hyphal tip isolations with be performed to ensure single genotypic isolate collection following protocol described by Powell et al. (2001). Subcultures of each isolate will be placed in -80oC long term storage following protocol described by Song et al. (2017). Dollar spot isolates from these same golf courses from 2010 well be revived from long term storage and grown on potato dextrose agar for future comparisons.ii)Fungicide sensitivity testing: Summer 2024 isolates will be incubated on PDA plates containing fungicides at a discriminatory dose including: the demethylase inhibitor fungicide propiconazole at 0.01 µg a.i. ml-1, the benzimidazole fungicide thiophanate methyl at 1,000 µg a.i. ml-1, and the succinate dehydrogenase inhibitor fungicide fluxapyroxad at 0.01 µg a.i. ml-1 (Popko et al. 2018). Five replications will be conducted for each fungal isolate. Following two days of incubation at 20oC, two measurements of fungal radial growth will be conducted and averaged for each isolate. Comparisons in relative growth to untreated control plates, as well as baseline isolates without known fungicide exposure will be conducted. Means will be statistically investigated using the mixed model procedure and the Tukey's HSD method within the R statistical package 'agricolae' (Mendiburu and Yaseen, 2020).iii) Comparative genomics of historically fungicide resistant populations: Genomic DNA from revived isolated sampled from Putman et al. (2010) and newly sampled isolates will be extracted using a CTAB extraction method (Zhang et al. 2023). DNA quality and quantity will be assessed using Nanodrop (Nanodrop Technologies, Waltham, MA) and Qubit™ Fluorometric quantitation (Thermo Fisher) Scientific, Inc., Waltham, MA, United States) respectively. Genomic DNA will be normalized and submitted to the University of Connecticut Center for Genome Innovation for restriction enzyme optimization and genotyping-by-sequencing run on a lane of Illumina NovaSeq 6000 (2x75bp) (Elshire et al. 2011). GBS variants will be aligned to previously generated C. jacksonii reference genome by Zheng et al. (2023). Resulting SNPs will be filtered based off minor allele frequency, read depth, and percent missing data using VCFtools (Danecek et al., 2011). Clonal lineages will be determined by generating pairwise identity by state (IBS) across all isolates collected and applying a 95% identity cutoff for clones.iv) Whole genome sequencing of representative dollar spot isolates: One isolate from each clonal lineage from Putman et al. and summer 2024 isolates will be submitted for whole genome sequencing. DNA will be extracted as described above and resulting DNA will be submitted to the University of Connecticut Center for Genome Innovation for TruSeq DNA Library preparation and sequenced on an Illumina NovaSeq 6000. De novo genome assemblies will be generated using Velvet v2.2.5 using default parameters (Zerbino and Birney 2008). Assembled genomes will used to identify regions of genome associated with clonal lineage differentiation utilizing GATK (Genome Analysis Toolkit).Objective 2a: Dollar spot epidemics within a single field are much more diverse genetically than previously supposedi)Hierarchal sampling of dollar spot foci within growing season: A 'Puttr' creeping bentgrass field maintained as a golf course fairway located at the University of Connecticut Plant Science Research Farm will be used for sample collection. This field has not been used for fungicide trials and is known to consistently develop heavy, natural dollar spot disease pressure. This field will be divided into four 4m x 4m quadrants where grid increments will be delineated at 1m (McDonald, 1997). When disease pressure is high (peak summer months when the Smith Kerns model exceeds 50% disease probability for 7 continuous days) one dollar spot isolate will be collected at the vertex of each grid, totaling 25 isolates per quadrant. C. jacksonii will be isolated in culture, aliquoted into -80 oC storage vessels, and DNA will be isolated and quantified as described above. ii) Genetic investigation dollar spot isolates within close proximity. Genomic DNA will be normalized and submitted to the University of Connecticut Center for Genome Innovation for Genotyping-by-sequencing run on a lane of Illumina NovaSeq 6000 (2x75bp) (Elshire et al. 2011). Resulting genetic variants will be parsed and filtered as described above. Additionally, one representative isolate from each clonal lineage will be submitted for Illumina NovaSeq 6000 whole genome sequencing and included in comparative genomic analysis as described above.Objective 2b: Seasonal weather shifts select for distinct pathogen populations within a field.i)Random sampling of dollar spot foci in Spring, Summer, and Fall: All non-fungicide trial creeping bentgrass research fields at the University of Connecticut Plant Science Research Farm will be used. Random sampling of dollar spot isolates will follow a zigzag pattern until 100 isolates have been collected. This sampling will occur on the onset of disease in May-June 2024, peak summer when the Smith Kerns model exceeds 50% disease probability for 7 continuous days (July-August), and conclusion of the growing season 14 days before the first predicted frost (September-October). Sampling will occur early in the morning to allow prioritization of symptomatic lesions with active mycelial growth typically observable between 5am-10am. C. jacksonii individuals will be isolated on antibiotic PDA as described above and placed in - 80oC storage. DNA will be extracted using a CTAB method as described above, however with the following modification: fungal tissue of 10 isolates will be weighted, normalized, then bulked together for extraction. This will be completed 10 times per season bulk to reduce the risk of sequencing bias towards overrepresented DNA samples in bulked DNA submission.ii) Sequencing and bulk segregant analysis: Quantity of total DNA within each bulked DNA extraction will be determined using Qubit™ Fluorometric quantitation (Thermo Fisher ) Scientific, Inc., Waltham, MA, United States) and 0.5 µg of DNA from each bulk will be subsampled to a master pool of DNA for each season. One bulk from Spring, Summer, and Fall will be submitted for Next Generation Bulked Segregant Analysis Sequencing using the Illumina HiSeq 2500 Platform (Song et al. 2017). Resulting reads will be aligned to the C. jacksonii reference genome (Zheng et al. 2023) using the BWA software (Langmead and Salzberg, 2012) and differential variants will be called using GATK (Genome Analysis Toolkit).