Stemphylium vesicarium (Wallr.) E.G. Simmons (Raghavendra Rao and Pavgi, 1975; Woudenberg et al., 2017) is a hemibiotrophic fungal pathogen and causal agent of Stemphylium leaf blight of onion (SLB) (Hay et al., 2021; Sharma and Sharma, 1999). SLB affects onions and other cultivated Allium spp. worldwide (Hay et al., 2021; Suheri and Price, 2000, 2001), including more recent reports from Brazil (de Souza Feitosa et al., 2023), Italy (Cortiello et al., 2023), Mauritius (Vally et al., 2024), Mexico (Reyes-Tena et al., 2024), New Zealand (Wright et al., 2019), Slovakia (Horáková et al., 2024), South Korea (Back et al., 2022), Taiwan (Wang et al., 2021) and the Ukraine (Klechkovskyi et al., 2023). S. vesicarium also affects other crops such as garlic (Suheri and Price, 2000), leek (Suheri and Price, 2001), asparagus (Graf et al., 2016), and pear (Köhl et al., 2009).

Onion production represents an important component of primary production for New York (NY), comprising approximately 2,400 ha with an annual value of US$63M (USDA NASS, 2024). Most of the intensive onion production in NY occurs on high organic content histosol (muck soils) in Genesee/Orleans Counties (Elba region), and Wayne, Oswego and Orange Counties. The impact of SLB on onion production in northern parts of the U.S.A., and in eastern Canada has escalated in recent years, transforming it from a sporadic threat into a persistent challenge (Hay et al., 2021). Today, SLB is now the most important foliar disease of onion in NY, with epidemics leading to premature plant death and reductions in bulb weight (Hay et al., 2021; Hoepting, 2018; Paibomesai et al., 2012). The emergence of SLB in NY has been associated with the development of resistance to several single site-specific modes of action fungicides which makes disease management increasingly challenging (Hay et al., 2019, 2022a; Pethybridge et al., 2016). Symptoms of SLB begin as water-soaked and pale brown to tan, mottled, oval to spindle-shaped, small lesions (Hay et al., 2021). Older lesions are darker gray or olive-brown in color due to profuse conidial production. As the disease develops, SLB lesions rapidly coalesce and develop into dieback of leaves leading to defoliation (Basallote-Ureba et al., 1999; Hay et al., 2021).

SLB epidemics may be initiated by multiple primary inoculum sources including overwintering inoculum as mycelia, conidia, or pseudothecia and ascospores on infested onion tissue from the previous season, alternative hosts such as weeds (Hay et al., 2021, 2022a, 2022b; McDonald et al., 2023; Stricker, 2021), infected seed (Aveling et al., 1993; Stricker, 2021), volunteer onions surviving from the previous season, or bare-root transplants (Hay et al., 2022b). Secondary spread of SLB within the cropping season results from multiple infection cycles typical of polycyclic disease epidemics (Hay et al., 2022b) resulting from rain and wind-blown dispersal of conidia (Gossen et al., 2021) or onion thrips mediated dispersal (Leach et al., 2020).

Population biology tools offer the opportunity to gather valuable insights into critical aspects of plant disease dynamics, including inoculum sources (Prussin et al., 2013; Zwankhuizen et al., 2000), migration trends (Goss, 2015), phylogeography of the pathogen (Linde et al., 2010), genetic diversity (Rieux et al., 2011), and modes of reproduction (Maciel et al., 2014; Rieux et al., 2011). Understanding the population genetic structure of a pathogen is essential for identifying factors that drive variability including mutations, mating systems, migration, population size, and natural selection (McDonald and Linde, 2002; Milgroom, 2015). Temporal analysis of populations provides insights into evolution and the presence of any genetic bottlenecks, expansions, or shifts in allele frequencies that may occur between growing seasons (Stauber et al., 2022). Spatial analysis can facilitate the identification of geographic structuring and potential barriers to gene flow among regions (Matsuda et al., 2015; Rampersad, 2021). By assessing the genetic diversity and population structure of plant pathogens, effective disease management strategies can be developed, as genetic composition can influence pathogenicity, virulence, and adaptation to environmental conditions (Çelik Oğuz and Karakaya, 2021; McDonald, 2015). Information surrounding the structure of plant pathogen populations is therefore essential to underpin the development of durable integrated disease management strategies to minimize crop loss.

Simple Sequence Repeats (SSRs) or microsatellite markers are a well-established tool to examine the population structure of plant pathogens due to their high polymorphism, co-dominant inheritance, and reproducibility (Jarne and Lagoda, 1996). Their ease of use, low DNA requirements, and affordability make them ideal for studying fine-scale population genetics (Selkoe and Toonen, 2006). Thus, SSR markers have been broadly used to assess genetic diversity, understand population structure, and monitor changes over time and space in pathogen populations (Moges et al., 2016; Rouxel et al., 2012; Widmark et al., 2011). A recent study developed nine SSR markers for S. vesicarium, providing essential molecular tools for population genetic studies (Heck et al., 2023). This study utilizes these SSR markers to investigate the spatiotemporal genetic diversity and population structure of S. vesicarium across geographically distinct onion production regions in NY over multiple years. By analyzing a larger and more geographically diverse set of isolates, this study aims to uncover patterns of genetic differentiation, admixture, and contribute to a deeper understanding of pathogen persistence and dispersal in agroecosystems, offering valuable information for the development of long-term, sustainable disease management strategies.

Stemphylium vesicarium populations from NY onion fields exhibited high genetic diversity with minimal spatial and temporal genetic structure. The analysis of nine SSR loci identified 158 distinct MLGs (75%) of 210 isolates, indicating high genotypic richness. The temporal fluctuations in eMLGs and genetic diversity indices suggested shifts in population structure potentially driven by environmental pressures or management practices. For example, the 2016 isolates tended to form a distinct genetic cluster indicating some divergence. Moreover, the observed admixture in S. vesicarium populations from 2018, 2020, and 2022, suggests ongoing gene flow and genetic mixing over time.

Similarly, the spatial distribution of eMLGs, along with comparatively high genetic diversity, particularly in Wayne and Oswego, suggest localized adaptation, where populations may be evolving at a different rate and/or in response to distinct agricultural practices in these regions. Although population structure identified fewer clusters (K = 2) than the five spatial populations analyzed, significant structuring was not present. Based on multiple population structure analysis, the Orange population showed some degree of genetic isolation with almost no admixture, while all other populations showed significant admixture, indicative of common source or gene flow among regions. Furthermore, UPGMA clustering grouped Elba and Finger Lake populations, and Oswego and Wayne populations within an additional distinct cluster. The Elba and Finger Lakes regions are geographically closer to each other but have distinctly different management strategies. The Finger Lakes region mostly consists of geographically isolated, small-scale, organic or low-input farms, on mineral soils. Conversely, the Elba region is composed of several large-scale conventional farms located on a pocket of histosol soil using high inputs and with fields located near each other. Wayne and Oswego counties are closer to each other, and differentiate from Elba, by having farms located on several smaller and geographically separate areas of histosoils. Orange County production is akin to Elba with multiple onion fields and farms neighboring each other, in a single, large area of histosols soil, and geographically distant from other regions in the study. S. vesicarium populations analyzed using amplified fragment length polymorphism markers also showed no evidence of spatial association between genotypes (Köhl et al., 2009).

The genetic variability in S. vesicarium populations from NY onion fields was therefore predominantly attributed to within populations, with minimal differentiation between populations. The 2016 population exhibited slightly significantly higher differentiation (Jost’s D = 0.087–0.098), likely to reflect initial genetic variation homogenized through gene flow or the influence of agronomic selection pressures (i.e., fungicide applications) in the subsequent years. The Orange population displayed a marginally higher significant Jost’s D value (0.10–0.23), suggesting localized genetic variation potentially driven by unique environmental conditions or specific management practices in that area. Low genetic differentiation implies that there is likely high gene flow among the populations, which can homogenize their genetic composition over time, possibly due to frequent migration events or evolutionary processes such as random genetic drift and mutation rates (Adhikari et al., 2021; Zhan, 2016).

Linkage disequilibrium analysis revealed significant non-random associations of alleles across loci in all populations except the Finger Lakes, which includes more organic fields compared to other regions, suggesting predominantly clonal reproduction. The reproductive biology of S. vesicarium is characterized by homothallism allowing self and outcrossing (Inderbitzin et al., 2005). Homothallic species like S. vesicarium can undergo syngamy between genetically identical haploid cells, resulting in haploid selfing and producing a population structure akin to clonal reproduction (Billiard et al., 2012). Conversely, outcrossing during sexual reproduction generates a recombining population structure (Milgroom, 1996; Smith et al., 1993; Tibayrenc et al., 1991). However, it remains unclear whether clonal genotypes arise solely from selfing, asexual reproduction, or a combination. While analyzing ascospore populations could help clarify the contribution of sexual reproduction, the homothallic nature of the pathogen makes controlled crosses challenging, leaving the role of outcrossing in natural populations unresolved (Attanayake et al., 2014). The non-significant linkage disequilibrium in the Finger Lakes population may suggest higher recombination, but its smaller sample size warrants caution in interpretation. Outcrossing through sexual reproduction is often linked to high genetic diversity due to recombination and the generation of novel genotypes (Atallah et al., 2004; Bihon et al., 2012; Brewer et al., 2015; McDonald, 1997; Milgroom, 1996). The significant linkage disequilibrium in populations with high genetic diversity in this study may seem counterintuitive. However, studies have shown that both in homothallic (Brewer et al., 2015) and heterothallic (Gañán-Betancur et al., 2021) pathogens, significant linkage disequilibrium can occur alongside high genetic diversity. Moreover, sampling timing could influence linkage disequilibrium interpretation. Samples in this study were collected later in the season when approaching harvest, when polycyclic asexual reproduction dominates and could bias results toward clonal structures of the population. Future studies should consider sampling at different time points in the growing season to better capture the full spectrum of reproductive modes of S. vesicarium.

The high genetic diversity of S. vesicarium may be attributed to factors such as importation of infected transplants from other states, and long-range dispersal of ascospores from pseudothecia produced in overwintering substrates like infested plant debris, alternative hosts including weeds, or volunteer onion plants (Gossen et al., 2021; Hay et al., 2021; McDonald et al., 2023). The significant role of external inoculum sources in SLB spread, suggested by the lack of significant spatiotemporal association (Hay et al., 2022b), likely drives the emergence of new genotypes, thereby sustaining continuous genetic diversity without clearly defined genetic structure. The high genotypic diversity may result from multiple introductions and subsequent population admixture, as supported by the population structure analyses. A similar pattern was observed in Alternaria brassicicola where multiple founder populations, resulting in the admixture, drove high gene and genotypic diversity (Linde et al., 2010). Additionally, high dispersal potential through airborne spores (Basallote-Ureba et al., 1999; Gossen et al., 2021; Katoch and Kumar, 2017; Simmons, 1969), insects (Leach et al., 2020), and the movement of infected planting material may facilitate genetic mixing over time and space. The lack of strong genetic structure implies uniform adaptation of the pathogen across locations and years, complicating management strategies.

Additional population genetic analyses, including S. vesicarium populations from other onion-growing regions in the U.S. and worldwide, could shed light on the pathogen’s dispersal patterns and invasion pathways. The coexistence of organic and conventional farms and varying host susceptibility levels, also likely influence the evolutionary dynamics of S. vesicarium. Future studies integrating host cultivar information, inoculum sources, and the role of infested onion transplants are crucial for a comprehensive understanding of genetic diversity and patterns. Overall, these findings highlight the need for integrated management strategies that account for regional genetic dynamics and monitoring genetic changes over time to effectively address pathogen adaptation and dispersal. Addressing the unresolved questions about the pathogen’s reproductive strategies and dispersal mechanisms will be key to mitigating the impact of S. vesicarium on onion production. This study provides valuable insights into the population biology of S. vesicarium, an economically important foliar pathogen affecting onion production in NY. Future studies should integrate advanced genomic tools, epidemiological data, and S. vesicarium populations from diverse regions, while considering management factors, reproductive strategies, and dispersal mechanisms, to better understand the pathogen’s evolution and inform comprehensive, adaptive disease management strategies.