Determining the relative importance of primary inoculum sources for Stemphylium leaf blight of onion in New York

1. To determine genetic diversity within and among S. vesicarium populations: 

1.1. Sample collection. Isolates of S. vesicarium obtained in 2022 and 2023 from onion leaf samples showing SLB symptoms prior to lodging, volunteers, and transplants were used in this study. These isolates are part of the permanent collection of more than 3,500 S. vesicarium isolates in the EVADE lab at Cornell AgriTech. Pathogen isolation was performed following a modified protocol described by Hay et al. (2019). Leaf lesions were examined to confirm the production of S. vesicarium conidia with a stereo microscope. Subsequently, 15 μL of 0.01% (v/v) Tween 20 (Sigma-Aldrich, St. Louis, MO) in sterilized distilled water were placed onto a sporulating lesion with the aid of a micropipette. The conidial suspension was then drawn from the sporulating lesion, placed onto 2 % water agar (WA; Hardy Diagnostics, Santa Maria, CA) amended with ampicillin (25 mg/L) (Fisher Scientific, Pittsburgh, PA) and spread by sloping the plate. After 5 h, germinating conidia were transferred as single spore cultures onto new Petri plates containing V8 juice agar amended with streptomycin (200 mg/liter). Finally, the monoconidial isolates were incubated at room temperature (25 ± 2°C) under conditions of 12 h light/dark cycle for 7 days. Pathogen confirmation was initially based on the morphology of conidia and fungal structures observed under a light microscope (40×; Miller and Schwartz 2008). For long-term preservation, single-conidial isolates were grown on synthetic low-nutrient agar (SNA) (Gerlach and Nirenberg 1982) and then agar plugs colonized with mycelia were placed in 1.5-mL tubes (Fisher Scientific, Pittsburgh, PA) containing sterile distilled water and kept at room temperature (25 ± 2°C) (Castellani 1963). Monoconidial isolates were also preserved in glycerol 30% and stored at -78ºC (± 2 ºC) (Heckly 1978). 

1.2. DNA extraction. Isolates were retrieved from long-term storage and grown on Petri plates containing V8 juice agar amended with streptomycin (200 mg/L) to produce mycelia. Plates were incubated at room temperature (25 ± 2°C) under conditions of 12 h light/dark cycle for 7 days. Mycelia was harvested by scraping the agar surface with a sterile scalpel and transferred into sterile foil to be dried overnight. Dried mycelia was used subsequently for DNA extraction protocols. Genomic DNA was extracted with the Wizard Extraction Kit (Promega Corp, Madison, WI) following the manufacturer’s recommendations. The specific primers KES1999 and KES2000, previously designed by Graf et al. (2015), are being used to corroborate the identity of the S. vesicarium isolates used in this study. Sequencing will be performed at Cornell Genomics Facility.

1.3. Microsatellites data. For the population genetics study, nine microsatellite markers developed by Heck et al. (2023; from Pethybridge Lab) will be used to characterize the genotypic diversity of the S. vesicarium populations collected in 2022 and 2023. Originally, Heck et al. (2023) tested 26 microsatellite markers on a subset of six S. vesicarium isolates collected from NY onion fields in 2016 and 2018 and amplified regions were sequenced to confirm the presence of the repeat motif of interest. Subsequently, a final set of nine microsatellites were selected, and primers were prepared with fluorescent dyes for polymorphism screening and multiplexing (Heck et al. 2023). In the present study, alleles at each of the nine microsatellite loci will be amplified from each monoconidial isolate using the primer sets designed by Heck et al. (2023; Table 1). Polymerase Chain Reaction (PCR) amplifications will be carried out in a C1000 Touch TM Thermal Cycler (Bio-Rad, Hercules, CA) using the nine primer sets, the genomic DNA obtained as previously described, and the Multiplex Master mix (Bioline, London, United Kingdom) in accordance with the manufacturer’s recommendations in a final PCR reaction volume of 12.5 μL. The PCR conditions will include an initial denaturation for 5 min at 95ºC, followed by 35 cycles of denaturation at 95ºC for 30 s, annealing at 57ºC for 30 s, an extension at 68ºC for 30 s, and a final extension at 68ºC for 5 min (Heck et al. 2023). PCR products will be sent for fragment analysis at the Cornell University, Institute of Biotechnology, Genomic Diversity Facility, using a GeneScan-500 LIZ size standard (Applied Biosystems). Chromatograms will be analyzed using Geneious Prime 2022.0.1 with the Microsatellite 1.4.7 plugin (Kearse et al. 2012).

1.4. Genetic diversity analysis: To study the genetic diversity of our populations, the number of multilocus genotypes (MLGs) will be determined for each population based on the size of the alleles at each of the nine loci. Once the MLG for each isolate is determined, MLGs frequencies will be calculated, and Nei’s index (H_exp) of genetic diversity will be assessed at each locus and population. Additional diversity indices will be calculated for each population including Evenness (E), Shannon-Wiener (H’), and Stoddart and Taylor’s (G’) as a measure of genotypic richness (Grünwald et al. 2003). Clonal fraction will also be calculated as 1- (MLG/N (total number of individuals per population)) to define the proportion of isolates in the population potentially originating from asexual reproduction (Zhan et al. 2003). The number of individuals per population (N), number of multilocus genotypes (MLGs), and Nei’s unbiased gene diversity index (H_exp) will be calculated with the poppr function in the poppr package v. 2.9.3 using R studio (Kamvar et al. 2014). Diversity indices of Shannon-Wiener (H’), Stoddart and Taylor’s (G’), Simpson (λ), and Evenness (E) will be calculated using the diversity_ci function in the poppr package with the argument ‘rarefy = TRUE’. To test for linkage disequilibrium and possible recombination in the populations, the index of association (I_A) and the standardized index of association (r_d) will be estimated with 999 permutations using the I_A function in the poppr P < 0.001 values will indicate deviation from the null hypothesis of no linkage disequilibrium. Analyses conducted with and without clone correction are also important when analyzing pathogen populations that reproduce clonally. Clone correction will be used to select for one individual representing each unique MLG per population.

2. To determine the population structure of S. vesicarium in NY onion fields.

2.1. Population biology studies involving indirect estimates of migration such as population structure (genetic differentiation) can also help us to understand the pattern of genetic variation within and among populations and whether migration is active (Milgroom 2015). In this study, population differentiation summary statistics such as index GST, Minimum Spanning Networks (MSN) and Principal Component Analysis (PCA) will be used to evaluate the population structure of S. vesicarium populations collected from NY onion crops. Population structure will be quantified using analysis of molecular variance (AMOVA) with the poppr.amova function in the poppr package (Kamvar et al. 2014). MSN will be calculated using Bruvo’s distance (Bruvo et al. 2004) with the function bruvo.msn in the poppr package. Discriminant Analysis of Principal Components (DAPC) will be conducted using the dapc function in the adegenet package in R studio (Jombart 2008). 

3. Pitfalls and limitations. 

3.1. If after clone correction, the sample size for each population decreases such that indices cannot be calculated, the entire set of samples collected from NY would be accounted as a single population and indices would be calculated for the NY population. Genotype-by-sequencing (GBS) can be used as an alternative to microsatellites for genetic diversity studies. GBS offers higher resolution genotyping at a relatively low-cost as it screens the entire genome but as a reduced-representation approach to whole-genome sequencing (Friel et al. 2021). GBS deploys a set of restriction enzymes to reduce the complexity of the genome facilitating the construction of GBS libraries. GBS is highly accurate and makes it possible to reach regions of the genome that are not accessible by using other sequencing approaches (He et al. 2014).