Cirsium arvense (L.) Scop. (Canada thistle) is a problematic weed that causes large economic losses in agriculture, driving the need for integrated weed management tools that include biological control agents (Orloff et al., 2018). Cirsium arvense can be found throughout temperate climates of the world, where it exists as a perennial herb that reproduces through an extensive rhizomatous root system and wind dispersed seeds (Tiley, 2010). Clonal rhizomes make C. arvense resilient to disturbance, particularly in tilled organic cropping systems that do not use synthetic herbicides for weed management (Moore, 1975). Organic producers in the Northern Great Plains region of the United States generally depend on tillage as a primary weed management tool, however this practice increases soil erosion due to wind and water and depletes soil organic matter over time (Lenhoff et al., 2017). Additionally, tillage can disperse vigorous C. arvense rhizomes, causing a rapid increase of the weed’s population (Tiley, 2010). As a result, C. arvense has become a serious management problem within organic cropping systems, where alternative management tools need to be explored (Tautges et al., 2016; Orloff et al., 2018).

The use of competitive annual crops is another common approach used to manage weeds in organic cropping systems (Bullock, 1992; Liebman and Dyck, 1993). Competitive crops can disrupt weed growth by reducing resource availability and niche dominance of weed species (Liebman and Dyck, 1993). However, the difficult nature of reducing C. arvense rhizomes, particularly in organic agriculture (Tautges et al., 2016; Orloff et al., 2018), has led to a search for alternative and integrated tactics, including biocontrol agents that inhibit root development (Berner et al., 2013; Cripps et al., 2014). The use of biocontrol agents can be challenging due to a lack of host specificity, varied responses to environmental conditions, and mismanagement. However, continued exploration of biocontrols for C. arvense has the potential to yield low-cost, long-term, host-specific options that can be integrated into existing weed management toolsets (Berner et al., 2013).

Puccinia punctiformis (F. Strauss) Rohl. (thistle rust) is a heterotrophic fungal pathogen of C. arvense that acts as a long-term systemic parasite (Buller, 1950; Menzies, 1953; Berner et al., 2013; Kentjens et al., 2023). As a parasite that consumes resources and weakens the root structure (Buller, 1950; Menzies, 1953), P. punctiformis is specific to C. arvense (Berner et al., 2013; Kentjens et al., 2023) and has been identified in temperate habitats around the globe (Berner et al., 2013; Kentjens et al., 2023). Once established in the roots, infected C. arvense can develop chlorotic leaf tissue with lesions, elongated stems, and growth irregularities which can reduce fitness and cause death (Buller, 1950; Berner et al., 2013). Diseased stems act as aboveground carriers for P. punctiformis spores, appearing as orange to dark-red pustules on leaves, where the fungus completes most of its five-stage heterothallic life cycle during summer months, eventually producing transmissible teliospores (Buller, 1950; Menzies, 1953; Kentjens et al., 2023). Teliospore-bearing thistle leaves senesce and abscise as precipitation and temperatures decline, where they can contact healthy C. arvense rosettes through wind or mechanical dispersion, leading to long-term systemic infection in new C. arvense hosts under ideal environmental conditions (French and Lightfield, 1990; Berner et al., 2013).

Puccinia punctiformis’ impact on C. arvense abundance has been well documented (French, 1990; Thomas et al., 1994; Berner et al., 2013; Cripps et al., 2014; Kentjens et al., 2023). However, to our knowledge, the effects of integrating the P. punctiformis biocontrol with a competitive crop sequence on C. arvense growth have not been studied. We addressed this gap in knowledge using greenhouse experiment, which assessed the impact of P. punctiformis on C. arvense growth and competitiveness. Specifically, our questions were: 1) What is the probability of observing P. punctiformis infected C. arvense over time, and does the percentage of infected C. arvense stems increase over time? 2) How does P. punctiformis affect C. arvense above- and belowground biomass, and does crop competition interact with the effects? 3) Using a relative competition intensity index (RCI), is the competitive ability of C. arvense reduced when P. punctiformis is integrated into a sequence of competitive annual crops? We hypothesized that the integration of P. punctiformis with a competitive crop sequence would lead to a significant reduction in above- and belowground C. arvense biomass, compared to individual effects from P. punctiformis or crop competition when used alone.

Sustainable C. arvense management in organic cropping systems is a primary challenge in temperate regions around the globe. Integrated weed management strategies are needed to reduce the abundance, slow the spread, and minimize the impact of C. arvense in cropping systems over a long term (Liebman et al., 2001; Liebman and Davis, 2009; Davis et al., 2018; Orloff et al., 2018). In this study, we found that the integration of P. punctiformis and crop competition interacted to impact C. arvense biomass and competitive ability. Integrated weed management of C. arvense that combines the P. punctiformis biocontrol with crop competition can reduce C. arvense vigor but requires careful consideration for effective use within complex cropping systems.

Repeated inoculations of C. arvense rosettes with P. punctiformis yielded systemically infected C. arvense stems in all phases of the crop sequence. Inoculation of rosettes resulted in few systemically infected C. arvense stems in the first phase (3-4 months of growth) of the crop sequence, but incidence of infection increased over time. The slow development of systemically infected stems is consistent with the general development of plant pathogens, which often require an incubation period before infected plants develop symptoms (Agrios, 2005). Our findings are also consistent with literature that suggests that P. punctiformis mostly resides asymptomatically within C. arvense rhizomes (Bailiss and Wilson, 1969), especially during the initial stages of infection. In a study testing asymptomatic C. arvense rosettes in proximity to P. punctiformis inoculations, Berner et al. (2015) discovered that up to 60% of asymptomatic rosettes were positive hosts for P. punctiformis. Therefore, the success of our inoculations was likely greater than what was observed aboveground.

While systemically infected stems were observed in most inoculated greenhouse pots, the majority of stems produced were asymptomatic. This supports the conclusion that P. punctiformis is primarily a root pathogen (Berner et al., 2015; Kentjens et al., 2023) that remains latent until adequate resources are gathered from the host and environmental conditions are suitable for the emergence of spore bearing C. arvense stems (Mendgen and Hahn, 2002). The stabilization of infected C. arvense stems after the fallow phase reflects the host’s capacity to support P. punctiformis, given the limitations of plant growth in greenhouse pots. Berner et al. (2015) and Watson and Koegh (1980) suggested that the robustness of infected C. arvense can be a factor that influences the development of systemically infected C. arvense stems, where a robust host is more likely to produce a relatively high abundance of infected stems, and systemic infection in a weaker host could produce fewer infected stems. It was concluded that systemic infection in a less robust host remains mostly asymptomatic and caused death more quickly than systemic infection in a robust host.

Cirsium arvense that was inoculated with the P. punctiformis biocontrol produced less belowground biomass compared to C. arvense that was not inoculated. Our results agree with the findings of Thomas et al.’s (1994) greenhouse experiment, where P. punctiformis inoculated C. arvense produced less root biomass than non-inoculated C. arvense. A weakened root system can directly impact aboveground biomass production, where root resources that would otherwise promote stem growth, are instead allocated to costly defense compounds, or become parasitized by P. punctiformis (Herms and Mattson, 1992; Thomas et al., 1994; Monson et al., 2022). This was demonstrated in our findings, where P. punctiformis inoculations yielded less aboveground biomass compared to C. arvense that was not inoculated, confirming that P. punctiformis inoculations can effectively impact the overall growth of C. arvense.

Competition with annual crops affected C. arvense aboveground growth, although the effects differed between crop species. Unexpectedly, peas were the most competitive annual crop species in the sequence, despite their relatively slow germination, shallow rooting depth, and open canopy (McKay et al., 2003). It is possible that wheat, a moderately competitive cereal species (Mason and Spaner, 2006), had a lasting impact on C. arvense vigor that wasn’t evident until the following pea phase. The weak competitive qualities of peas may have facilitated a recovery in C. arvense vigor, becoming evident in the following phase, where safflower had the lowest relative competition intensity. However, safflower, known to be a weak competitor in the early stages of growth (Emongor and Oagile, 2017), was disadvantaged as the last crop in the sequence. It is possible that greenhouse pots with fully developed roots gave C. arvense a strong competitive advantage by the final phase of the crop sequence; seeding safflower directly into a dense and confined C. arvense root network likely impacted optimal safflower development.

When inoculated C. arvense was grown in mixed pots with interspecific crop competition, the biocontrol interacted additively with crop competition to further reduce above-and belowground biomass, more than individual impacts from the biocontrol or crop competition alone. Although C. arvense was never eradicated by the combination of P. punctiformis and crop competition, there was an interaction between the crop phases and the inoculation treatments, where the difference between the P. punctiformis inoculated and the non-inoculated (control) relative competition intensities gradually increased as the crop sequence progressed. As P. punctiformis inoculations did not immediately affect C. arvense’s competitive ability, but increased through time, the effects appear to be associated with the establishment of infected C. arvense stems. The greatest impact on C. arvense competition emerged after aboveground disease incidence stabilized and persisted through time.

Although we didn’t evaluate physiological responses of C. arvense, there is potential to accelerate disease establishment and increase the severity of P. punctiformis infection by stimulating hormonal responses (Clark et al., 2020), thus enhancing future integrations of the biocontrol. Overall, these results support our hypothesis and provide evidence in favor of integrated weed management as an effective strategy for C. arvense control (Demers et al., 2006; Liebman and Davis, 2009; Sciegienka et al., 2011; Davis et al., 2018; Orloff et al., 2018).

While crop competition is already a common integrated weed management practice (Pavlychenko and Harrington, 1934; Bullock, 1992; Liebman and Dyck, 1993; Liebman and Davis, 2009), there remain practical challenges to the integration of the P. punctiformis biocontrol in field settings. Inoculum sourcing and mass production is limited by the inability to culture transmissible teliospores (Kentjens et al., 2023), creating a reliance on the harvest of teliospore bearing C. arvense. Limitations in inoculum ultimately reduce the scalability of the biocontrol under current sourcing methods. Most natural transmissions of P. punctiformis are limited to 12 meters from the source plant, with no transmissions occurring beyond 17 meters (Berner et al., 2015). Insect vectors or mowing have shown potential to transmit P. punctiformis and increase infection levels across fields (Demers et al., 2006; Wandeler and Bacher, 2006), however, careful cropping system management is required to facilitate effective spore distributions. The greenhouse environment simplifies biocontrol manipulations, but successful integration of P. punctiformis in a field setting will be dependent on variable environmental conditions and cropping system management that can influence survivability and germination of the biocontrol (French and Lightfield, 1990; Berner et al., 2013; Kentjens et al., 2023). Additionally, Thomas et al. (1994) found that P. punctiformis inoculations did not impact aboveground biomass production compared to non-inoculated C. arvense, suggesting inconsistent performance of the pathogen. Inconsistencies in the biocontrol’s impact on C. arvense aboveground growth may be an indication of genetic variability within the host and pathogen populations, where disease severity can be determined by a range of resistance mechanisms in C. arvense or virulence factors in P. punctiformis. Despite inconsistencies and challenges, P. punctiformis has shown potential to increase C. arvense’s vulnerability to integrated weed management tactics, making the biocontrol a viable management option.

The fungal biocontrol, P. punctiformis can be successfully integrated with crop competition as a C. arvense management tool. In this greenhouse study, inoculation of C. arvense rosettes with P. punctiformis teliospores caused an increase of symptomatically infected C. arvense stems over time, impacting above- and belowground C. arvense biomass production. Furthermore, P. punctiformis intensified the effects of crop competition when the biocontrol was integrated into a simulated crop sequence. While the use of P. punctiformis is possible in a greenhouse, successful integration of the biocontrol into a field setting will be dependent on a combination of environmental factors and deliberate cropping system management. Puccinia punctiformis is not a singular management solution for C. arvense, however it has potential to be integrated as a low-cost, and low-input biocontrol agent that can improve sustainable management of C. arvense.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

DC was the primary author, who conducted the data collection, statistical analysis, and writing. CL assisted with experimental design, statistical analysis, and editing. JE wrote the initial grant proposal and edited the manuscript. FM guided the writing and edited the manuscript. TS supervised data collection, assisted with statistical analysis, guided the writing, and edited the manuscript. All authors contributed to the article and approved the submitted version.